Processes for intermediates for macrocyclic compounds

ABSTRACT

The present invention is directed to novel macrocyclic compounds of formula (I) and their pharmaceutically acceptable salts, hydrates or solvates: 
                         
wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , n 1 , m, p Z 1 , Z 2 , and Z 3  are as describe in the specification. The invention also relates to compounds of formula (I) which are antagonists of the motilin receptor and are useful in the treatment of disorders associated with this receptor and with or with motility dysfunction.

RELATED APPLICATION INFORMATION

This application is a continuation under 35 U.S.C. §120 of U.S. patentapplication Ser. No. 10/872,142, filed Jun. 18, 2004, now U.S. Pat. No.7,521,420, which claims the benefit of U.S. Patent Application Ser. No.60/479,223, filed Jun. 18, 2003. The disclosure of each application isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel conformationally-definedmacrocyclic compounds, pharmaceutical compositions comprising same andintermediates used in their manufacture. More particularly, theinvention relates to macrocyclic compounds that have been demonstratedto selectively antagonize the activity of the motilin receptor. Theinvention further relates to macrocyclic compounds useful astherapeutics for a range of gastrointestinal disorders, in particularthose in which malfunction of gastric motility or increased motilinsecretion is observed, such as hypermotilinemia, irritable bowelsyndrome and dyspepsia.

BACKGROUND OF THE INVENTION

A number of peptide hormones are involved in the control of thedifferent functions in the gastrointestinal (GI) tract, includingabsorption, secretion, blood flow and motility (Mulvihill, et al. inBasic and Clinical Endocrinology, 4^(th) edition, Greenspan, F. S.;Baxter, J. D., eds., Appleton & Lange: Norwalk, Conn., 1994, pp551-570). Since interactions between the brain and GI system arecritical to the proper modulation of these functions, these peptides canbe produced locally in the GI tract or distally in the CNS.

One of these peptide hormones, motilin, a linear 22-amino acid peptide,plays a critical regulatory role in the GI physiological system thoughgoverning of fasting gastrointestinal motor activity. As such, thepeptide is periodically released from the duodenal mucosa during fastingin mammals, including humans. More precisely, motilin exerts a powerfuleffect on gastric motility through the contraction of gastrointestinalsmooth muscle to stimulate gastric emptying, decrease intestinal transittime and initiate phase III of the migrating motor complex in the smallbowel (Itoh, Z., Ed., Motilin, Academic Press: San Diego, Calif., 1990,ASIN: 0123757304; Nelson, D. K. Dig. Dis. Sci. 1996, 41, 2006-2015;Peeters, T. L.; Vantrappen, G.; Janssens, J. Gastroenterology 1980, 79,716-719).

Motilin exerts these effects through receptors located predominantly onthe human antrum and proximal duodenum, although its receptors are foundin other regions of the GI tract as well (Peeters, T. L.; Bormans, V.;Vantrappen, G. Regul. Pept. 1988, 23, 171-182). Therefore, motilinhormone is involved in motility of both the upper and lower parts of theGI system (Williams et al. Am. J. Physiol. 1992, 262, G50-G55). Inaddition, motilin and its receptors have been found in the CNS andperiphery, suggesting a physiological role in the nervous system thathas not yet been definitively elucidated (Depoortere, I.; Peeters, T. L.Am. J. Physiol. 1997, 272, G994-999 and O'Donohue, T. L et al. Peptides1981, 2, 467-477). For example, motilin receptors in the brain have beensuggested to play a regulatory role in a number of CNS functions,including feeding and drinking behavior, micturition reflex, central andbrain stem neuronal modulation and pituitary hormone secretion (Itoh, Z.Motilin and Clinical Applications. Peptides 1997, 18, 593-608; Asakawa,A.; Inui, A.; Momose, K.; et al., M. Peptides 1998, 19, 987-990 andRosenfeld, D. J.; Garthwaite, T. L. Physiol. Behav. 1987, 39, 753-756).Physiological studies have provided confirmatory evidence that motilincan indeed have an effect on feeding behavior (Rosenfeld, D. J.;Garthwaite, T. L. Phys. Behav. 1987, 39, 735-736).

The recent identification and cloning of the human motilin receptor (WO99/64436) has simplified and accelerated the search for agents which canmodulate its activity for specific therapeutic purposes.

Due to the critical and direct involvement of motilin in control ofgastric motility, agents that either diminish (hypomotility) or enhance(hypermotility) the activity at the motilin receptor, are a particularlyattractive area for further investigation in the search for neweffective pharmaceuticals towards these indications.

Peptidic agonists of the motilin receptor, which have clinicalapplication for the treatment of hypomotility disorders, have beenreported (U.S. Pat. Nos. 5,695,952; 5,721,353; 6,018,037; 6,380,158;6,420,521, U.S. Appl. 2001/0041791, WO 98/42840; WO 01/00830 and WO02/059141). Derivatives of erythromycin, commonly referred to asmotilides, have also been reported as agonists of the motilin receptor(U.S. Pat. Nos. 4,920,102; 5,008,249; 5,175,150; 5,418,224; 5,470,961;5,523,401, 5,554,605; 5,658,888; 5,854,407; 5,912,235; 6,100,239;6,165,985; 6,403,775).

Antagonists of the motilin receptor are potentially extremely useful astherapeutic treatments for diseases associated with hypermotility andhypermotilinemia, including irritable bowel syndrome, dyspepsia,gastroesophogeal reflux disorders, Crohn's disease, ulcerative colitis,pancreatitis, infantile hypertrophic pyloric stenosis, diabetesmellitus, obesity, malabsorption syndrome, carcinoid syndrome, diarrhea,atrophic colitis or gastritis, gastrointestinal dumping syndrome,postgastroenterectomy syndrome, gastric stasis and eating disordersleading to obesity.

A variety of peptidic compounds have been described as antagonists ofthe motilin receptor (Depoortere, I.; Macielag, M. J.; Galdes, A.;Peeters, T. L. Eur. J. Pharmacol. 1995, 286, 241-247; U.S. Pat. Nos.5,470,830; 6,255,285; 6,586,630; 6,720,433; U.S. 2003/0176643; WO02/64623). These peptidic antagonists suffer from the known limitationsof peptides as drug molecules, in particular poor oral bioavailabilityand degradative metabolism.

Cyclization of peptidic derivatives is a method employed to improve theproperties of a linear peptide both with respect to metabolic stabilityand conformational freedom. Cyclic molecules tend to be more resistantto metabolic enzymes. Such cyclic tetrapeptide motilin antagonists havebeen reported (Haramura, M. et al J. Med. Chem. 2002, 45, 670-675, U.S.2003/0191053; WO 02/16404).

Other motilin antagonists, which are non-peptidic and non-cyclic innature have also been reported (U.S. Pat. Nos. 5,972,939; 6,384,031;6,392,040; 6,423,714; 6,511,980; 6,624,165; 6,667,309; U.S.2002/0111484; 2001/041701; 2002/0103238; 2001/0056106, 2002/0013352;2003/0203906 and 2002/0002192)

The macrocyclic motilin antagonists of the present invention compriseelements of both peptidic and non-peptidic structures in a combinationwhich has not been pursued for this application previously.

Indeed, the structural features of antagonists of the present inventionare different. In particular, within the known motilin antagonists whichare cyclic peptides, it was found that such derivatives containingD-amino acids were devoid of activity. In contrast, for thetripeptidomimetic compounds of the present invention, theD-stereochemistry is required for two of the three building elements.

The motilin antagonists of the present invention are also distinct fromthe prior art in that they comprise a tether element to fulfill the dualrole of controlling conformations and providing additional sites forinteraction either through hydrophobic interactions, hydrogen bonding ordipole-dipole interactions.

SUMMARY OF THE INVENTION

In a first aspect, the present invention is directed to compounds offormula (I):

and pharmaceutically acceptable salts, hydrates or solvates thereofwherein:Z₁, Z₂ and Z₃ are independently selected from the group consisting of O,N and NR₁₀, wherein R₁₀ is selected from the group consisting ofhydrogen, lower alkyl, and substituted lower alkyl;R₁ is independently selected from the group consisting of lower alkylsubstituted with aryl, lower alkyl substituted with substituted aryl,lower alkyl substituted with heteroaryl and lower alkyl substituted withsubstituted heteroaryl;R₂ is hydrogen;R₃ is independently selected from the group consisting of alkyl andcycloalkyl with the proviso that when Z₁ is N, R₃ can form a four, five,six or seven-membered heterocyclic ring together with Z₁;R₄ is hydrogen;R₅ and R₆ are independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,heterocyclic, substituted heterocyclic, aryl, substituted aryl,heteroaryl and substituted heteroaryl, with the proviso that at leastone of R₅ and R₆ is hydrogen;X is selected from the group consisting of O, NR₈, and N(R₉)₂ ⁺;

-   -   wherein R₈ is selected from the group consisting of hydrogen,        lower alkyl, substituted lower alkyl, formyl, acyl,        carboxyalkyl, carboxyaryl, amido, sulfonyl, sulfonamido and        amidino; and    -   R₉ is selected from the group consisting of hydrogen, lower        alkyl, and substituted lower alkyl;        m, n₁ and p are independently selected from 0, 1 or 2; and        T is a bivalent radical of formula II:        —U—(CH₂)_(d)—W—Y—Z—(CH₂)_(e)—  (II)    -   wherein d and e are independently selected from 0, 1, 2, 3, 4 or        5;    -   wherein U is bonded to X of formula (I) and is —CH₂— or —C(═O)—;    -   wherein Y and Z are each optionally present;    -   W, Y and Z are independently selected from the group consisting        of: —O—, —NR₂₈—, —S—, —SO—, —SO₂—, —C(═O)—, —C(═O)—O—,        —O—C(═O)—, —C(═O)—NH—, —NH—C(═O)—, —SO₂—NH—, —NH—SO₂—,        —CR₂₉R₃₀—, —CH═CH— with a configuration Z or E, and —C≡C—, or        from a ring structure independently selected from the group:

-   -   wherein any carbon atom contained within said ring structure,        can be replaced by a nitrogen atom, with the proviso that if        said ring structure is a monocyclic ring structure, it does not        comprise more than four nitrogen atoms and if said ring        structure is a bicyclic ring structure, it does not comprise        more than six nitrogen atoms;    -   G₁ and G₂ each independently represent a covalent bond or a        bivalent radical selected from the group consisting of —O—,        —NR₄₁—, —S—, —SO—, —SO₂—, —C(═O)—, —C(═O)—O—, —O—C(═O)—,        —C(═O)NH—, —NH—C(═O)—, —SO₂—NH—, —NH—SO₂—, —CR₄₂R₄₃—, —CH═CH—        with a configuration Z or E, and —C≡C—; with the proviso that G₁        is bonded closer to U than G₂;    -   K₁, K₂, K₃, K₄, K₆, K₁₅ and K₁₆ are independently selected from        the group consisting of O, NR₄₄ and S;    -   f is selected from 1, 2, 3, 4, 5 or 6;    -   R₃₁, R₃₂, R₃₈, R₃₉, R₄₈ and R₄₉ are independently selected from        hydrogen, halogen, alkyl, substituted alkyl, cycloalkyl,        substituted cycloalkyl, heterocyclic, substituted heterocyclic,        aryl, substituted aryl, heteroaryl, substituted heteroaryl,        hydroxy, alkoxy, aryloxy, amino, halogen, formyl, acyl, carboxy,        carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido,        amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl and        sulfonamido; and    -   R₃₃, R₃₄, R₃₅, R₃₆, R₃₇, R₄₇, R₅₀ and R₅₁ are independently        selected from hydrogen, halogen, alkyl, substituted alkyl,        cycloalkyl, substituted cycloalkyl, heterocyclic, substituted        heterocyclic, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, halogen,        formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido,        carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto,        sulfinyl, sulfonyl and sulfonamido.

In a second aspect, the invention also proposes compounds of formula (1)which are antagonists of the motilin receptor.

In a third aspect, the invention proposes a method of treating adisorder associated with the motilin receptor or motility dysfunction inhumans and other mammals comprising administering a therapeuticallyeffective amount of a compound of formula (1).

While the invention will be described in conjunction with exampleembodiments, it will be understood that it is not intended to limit thescope of the invention to such embodiment. On the contrary, it isintended to cover all alternatives, modifications and equivalents as maybe included as defined by the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

Preferably in formula (I), as depicted hereinabove, R₁ is selected fromthe group consisting of —(CH₂)_(q)R₁₁, and —CHR₁₂R₁₃

-   -   wherein q is 0, 1, 2 or 3; and    -   R₁₁ and R₁₂ are independently selected from a ring structure        from the following group:

-   -   wherein any carbon atom in said ring structure can be replaced a        nitrogen atom, with the proviso that if said ring structure is a        monocyclic ring structure, it does not comprise more than four        nitrogen atoms and if said ring structure is a bicyclic ring        structure, it does not comprise more than six nitrogen atoms;    -   A₁, A₂, A₃, A₄ and A₅ are each optionally present and are        independently selected from the group consisting of halogen,        alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,        heterocyclic, substituted heterocyclic, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy,        amino, halogen, formyl, acyl, carboxy, carboxyalkyl,        carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino,        cyano, nitro, mercapto, sulfinyl, sulfonyl and sulfonamido;    -   B₁, B₂, B₃, and B₄ are independently selected from NR₁₄, S or O,        wherein R₁₄ is selected from the group consisting of hydrogen,        alkyl, substituted alkyl, formyl, acyl, carboxyalkyl,        carboxyaryl, amido, sulfonyl and sulfonamido;    -   R₁₃ is as defined for as R₁₁ and R₁₂ or is selected from the        group comprising lower alkyl, substituted lower alkyl, hydroxy,        alkoxy, aryloxy, amino, carboxy, carboxyalkyl, carboxyaryl, and        amido.        wherein A₁, A₂, A₃, A₄ and A₅ are most preferably selected from        halogen, trifluoromethyl, C₁₋₆ alkyl or C₁₋₆ alkoxy.

Preferably, R₁₁, R₁₂ and R₁₃ are selected from the group consisting of:

wherein R_(a) and R_(b) are chosen from the group consisting of Cl, F,CF₃, OCH₃, OH, and C(CH₃)₃ and CH₃.

Also preferably, R₃ in formula (I), is selected from the groupconsisting of:

-   -   —(CH₂)_(s)CH₃, —CH(CH₃)(CH₂)_(t)CH₃, —CH(OR₁₅)CH₃, —CH₂SCH₃        —CH₂CH₂SCH₃, —CH₂S(═O)CH₃, —CH₂CH₂S(═O)CH₃, —CH₂S(═O)₂CH₃,        —CH₂CH₂S(═O)₂CH₃, —(CH₂)_(u)CH(CH₃)₂, —C(CH₃)₃, and        —(CH₂)_(y)—R₂₁, wherein:        -   s and u are independently selected from 0, 1, 2, 3, 4 or 5;        -   t is independently selected from 1, 2, 3 or 4;        -   y is selected from 0, 1, 2, 3 or 4;        -   R₁₅ is selected from the group consisting of hydrogen,            alkyl, substituted alkyl, formyl and acyl;        -   R₂₁ is selected from a ring structure selected from the            following group:

-   -   -   wherein any carbon atom in said ring structure can be            replaced by a nitrogen atom, with the proviso that if said            ring structure is a monocyclic ring structure, it does not            comprise more than four nitrogen atoms and if said ring            structure is a bicyclic ring structure, it does not comprise            more than six nitrogen atoms;        -   z is selected from 1, 2, 3, 4 or 5;        -   E₁, E₂ and E₃ are each optionally present and are            independently selected from the group consisting of halogen,            alkyl, substituted alkyl, cycloalkyl, substituted            cycloalkyl, heterocyclic, substituted heterocyclic, aryl,            substituted aryl, heteroaryl, substituted heteroaryl,            hydroxy, alkoxy, aryloxy, amino, halogen, formyl, acyl,            carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl,            guanidino, ureido, amidino, cyano, nitro, mercapto,            sulfinyl, sulfonyl and sulfonamido; and        -   J is optionally present and is selected from the group            consisting of alkyl, substituted alkyl, cycloalkyl,            substituted cycloalkyl, heterocyclic, substituted            heterocyclic, aryl, substituted aryl, heteroaryl,            substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo,            amino, halogen, formyl, acyl, carboxy, carboxyalkyl,            carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino,            mercapto, sulfinyl, sulfonyl and sulfonamido.

The tether portion (T) of formula (I) is preferably selected from thegroup consisting of:

wherein L₁ is O, NH or NMe; L₂ is CH or N; L₃ is CH or N; L₄ is O orCH₂; L₅ is CH or N L₆ is CR₅₂R₅₃ or O; R₄₆ is H or CH₃;R₅₂, R₅₃, R₅₄, R₅₅, R₅₆ and R₅₇ are independently selected fromhydrogen, lower alkyl, substituted lower alkyl, hydroxy, alkoxy,aryloxy, amino, and oxo; or R₅₂ together with R₅₃ or R₅₄ together withR₅₅ or R₅₆ together with R₅₇ can independently form a three toseven-membered cyclic ring comprising carbon, oxygen, sulfur and/ornitrogen atoms;(X) is the site of a covalent bond to X in formula (I); and(Z₃) is the site of a covalent bond to Z₃ in formula (I).

In a particularly preferred embodiment of the invention, there areprovided compounds of formula (I) wherein m, n and p are 0, X, Z₁, Z₂and Z₃ are NH and R₂, R₄ and R₅ are hydrogen, represented by formula(III):

According to another aspect of the invention, there are providedcompounds of formula (I) wherein when Z₁ is a nitrogen atom, R₃ forms afour, five, six or seven-membered heterocyclic ring together with Z₁,represented by formula (IV):

wherein said heterocyclic ring may contain a second nitrogen atom, or anoxygen, or sulfur atom;n₂ is selected from 0, 1, 2 or 3R₇ is optionally present and is selected from the group consisting ofalkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,heterocyclic, substituted heterocyclic, aryl, substituted aryl,heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo,amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido,carbamoyl, guanidino, ureido, amidino, mercapto, sulfinyl, sulfonyl andsulfonamido.

It is to be understood, that in the context of the present invention,the terms amino, guanidine, ureido and amidino encompass substitutedderivatives thereof as well.

Preferably, the invention provides a method of treating a disorderassociated with hypermotility or hypermotilinemia in humans and othermammals comprising administering a therapeutically effective amount of acompound of formula (1).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts Scheme 1 presenting a general synthetic strategy toconformationally-defined macrocycles of the present invention.

FIG. 2 depicts the standard procedure for the synthesis of tether T8 ofExample 16.

FIG. 3 depicts the standard procedure for the synthesis of tether T9 ofExample 17.

FIG. 4 depicts the standard procedure for the synthesis ofDdz-propargylamine of Example 18.

FIG. 5A depicts the standard procedure for the synthesis of tether T10of Example 19.

FIG. 5B depicts the second synthetic route to tether T10 of Example 19.

FIG. 6 depicts the standard procedure for the synthesis of Tether T11 ofExample 20.

FIG. 7 depicts the standard procedure for the synthesis of tether T12 ofExample 26.

FIG. 8 depicts the procedure for synthesis of PPh₃-DIAD adduct ofExample 29-C.

FIG. 9 depicts the standard procedure for attachment of tethers viareductive amination of Example 30.

FIG. 10 depicts the standard procedure for the synthesis of tether T28of Example 32.

FIG. 11 the standard procedure for the synthesis of tether T32 ofExample 36.

FIGS. 12A, 12B depict the standard procedure for the synthesis of tetherT33a and T33b of Example 37.

FIG. 13 depicts the standard procedure for the synthesis of tether T34of Example 38.

FIG. 14 depicts the standard procedure for the synthesis of tether T35of Example 39.

FIG. 15 depicts the standard procedure for the synthesis of tether T36of Example 40.

FIG. 16 depicts the standard procedure for the synthesis of tether T37of Example 41.

FIG. 17 depicts the standard procedure for the synthesis of tether T38of Example 42.

Chiral T38 can be accessed through the use of asymmetric synthesismethods, resolution or chiral chromatography techniques available in theliterature.

HPLC (standard gradient) t_(R)=8.46 min

Chiral material can be accessed by starting with the chiral epoxide. Forexample, the (S)-isomer of T38 was constructed in 89% overall yield from(S)-propylene oxide.

FIG. 18 depicts the standard procedure for the synthesis of tether T39of Example 43.

Chiral T39 can be accessed through the use of asymmetric synthesismethods, resolution or chiral chromatography techniques available in theliterature.

FIG. 19 depicts the standard procedure for the synthesis of tether T40of Example 44.

Chiral T40 can be accessed through the use of asymmetric synthesismethods, resolution or chiral chromatography techniques available in theliterature.

FIG. 20 depicts the standard procedure for the synthesis of tether T41of Example 45.

FIG. 21 depicts the standard procedure for the synthesis of tether T42of Example 46.

FIG. 22 depicts Scheme 2 of the thioester strategy for macrocycliccompounds of the present invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

Although preferred embodiments of the present invention have beendescribed in detail herein and illustrated in the accompanyingstructures, schemes and tables, it is to be understood that theinvention is not limited to these precise embodiments and that variouschanges and modifications may be effected therein without departing fromthe scope or spirit of the present invention.

Specifically preferred compounds of the present invention, include, butare not limited to:

In addition to the preferred tethers (T) illustrated previously, otherspecific tethers employed for compounds of the invention are shownhereinbelow:

In a preferred embodiment, the present invention is directed to a methodof treating irritable bowel syndrome, dyspepsia, Crohn's disease,gastroesophogeal reflux disorders, ulcerative colitis, pancreatitis,infantile hypertrophic pyloric stenosis, carcinoid syndrome,malabsorption syndrome, diarrhea, diabetes mellitus, obesity,postgastroenterectomy syndrome, atrophic colitis or gastritis, gastricstasis, gastrointestinal dumping syndrome, celiac disease and eatingdisorders leading to obesity in humans and other mammals comprisingadministering a therapeutically effective amount of a compound offormula (I).

Synthetic Methods

A. General Information

-   -   Reagents and solvents were of reagent quality or better and were        used as obtained from various commercial suppliers unless        otherwise noted. DMF, DCM and THF used are of DriSolv® (EM        Science, now EMD Chemicals, Inc., part of Merck KgaA, Darmstadt,        Germany) or synthesis grade quality except for (i)        deprotection, (ii) resin capping reactions and (iii) washing.        NMP used for the amino acid (AA) coupling reactions is of        analytical grade. DMF was adequately degassed by placing under        vacuum for a minimum of 30 min prior to use. Tyr(3tBu) was        synthesized following the method reported in JP2000 44595. Cpa        was made using literature methods (Tetrahedron: Asymmetry 2003,        14, 3575-3580) or obtained commercially. Boc- and Fmoc-protected        amino acids and side chain protected derivatives, including        those of N-methyl and unnatural amino acids, were obtained from        commercial suppliers or synthesized through standard        methodologies known to those in the art. Ddz-amino acids were        either synthesized by standard procedures or obtained        commercially from Orpegen (Heidelberg, Germany) or Advanced        ChemTech (Louisville, Ky., USA). Bts-amino acids were        synthesized as described in Example 6. Hydroxy acids were        obtained from commercial suppliers or synthesized from the        corresponding amino acids by literature methods. Analytical TLC        was performed on pre-coated plates of silica gel 60F254 (0.25 mm        thickness) containing a fluorescent indicator. The term        “concentrated/evaporated under reduced pressure” indicates        evaporation utilizing a rotary evaporator under either water        aspirator pressure or the stronger vacuum provided by a        mechanical oil vacuum pump as appropriate for the solvent being        removed. “Dry pack” indicates chromatography on silica gel that        has not been pre-treated with solvent, generally applied on        larger scales for purifications where a large difference in        R_(f) exists between the desired product and any impurities. For        solid phase chemistry processes, “dried in the standard manner”        is that the resin is dried first in air (1 h), and subsequently        under vacuum (oil pump usually) until full dryness is attained        (˜30 min to O/N).        B. Synthetic Methods for Building Blocks of the Invention

Example 6 Standard Procedure for the Synthesis of Bts-Amino Acids

-   -   To a solution of the amino acid or amino acid derivative (0.1        mol, 1.0 eq) in 0.25 N sodium hydroxide (0.08 mol, 0.8 eq) with        an initial pH of approximately 9.5 (pH meter) at rt, solid        Bts-Cl (0.11 mol, 1.1 eq) was added in one portion. The        resulting suspension was stirred vigorously for 2-3 d. The pH of        the reaction should be adjusted with 5.0 N sodium hydroxide as        required to remain within the range 9.5-10.0 during this time.        Typically, the pH has to be adjusted every 20-30 min during the        first 5 h. Once the pH stops dropping, it is an indication that        the reaction is almost complete. This can be confirmed by TLC        (EtOAc:MeOH, 95:5). Upon completion, the reaction mixture was        washed with Et₂O. Washing is continued until the absence of        non-polar impurities in the aqueous layer is confirmed by TLC        (typically 3×100 mL). The aqueous solution was then cooled to 0°        C., acidified to pH 2.0 with 1 N HCl until no additional        cloudiness forms, and extracted with EtOAc (3×100 mL).        Alternatively, a mixture of DCM and EtOAc may be used as the        extraction solvent, depending on the solubility of the product        obtained from different amino acids or derivatives. Note that        DCM cannot be used solely as solvent because of the emulsion        formed during extraction. The combined organic phases were        washed with brine (2×150 mL), dried over MgSO₄, filtered and        evaporated under reduced pressure. DCM (1×) and hexanes (2×)        were evaporated from the residue in order to ensure complete        removal of the EtOAc and give the desired compound as a solid in        55-98% yield.    -   The following are modifications that have proven useful for        certain amino acids:    -   Gly, Ala, D-Ala, β-Ala and GABA: Use 1.5 eq of amino acid per eq        of Bts-Cl, in order to prevent dibetsylation.    -   Met: Carry out the reaction under N₂ to prevent oxidation.    -   Gln and Asn: Due to the solubility of Bts-Gln and Bts-Asn, the        work-up required is modified from the standard procedure: Upon        completion of the reaction, the reaction mixture was washed with        diethyl ether. Washing is continued until the absence of        non-polar impurities in the aqueous layer is confirmed by TLC        (typically 3×100 mL). The aqueous phase was then cooled to 0° C.        and acidified to pH 2.0 with 6 N HCl. 6 N HCl was employed to        minimize the volume of the solution due to the water solubility        of Bts-Gln and Bts-Asn. (They are, in contrast, difficult to        dissolve in DCM, EtOAc or chloroform.) The solution was        maintained at 0° C. for 10 min and the product was collected by        filtration as a white precipitate. The solid was washed with        cold water (1×), cold brine (2×) and water (1×, 25° C.). The pH        of this wash was taken, if it is not approximately 4, the solid        was washed again with water. Finally, the solid was washed with        cold EtOAc, then with cold Et₂O (2×), and finally dried under        vacuum (oil pump) (83-85% yield).        C. General Synthetic Strategy to Conformationally-Defined        Macrocycles of the Present Invention

The compounds of Formula I can be synthesized using traditional solutionsynthesis techniques or solid phase chemistry methods. In either, theconstruction involves four phases: first, synthesis of the buildingblocks, including one to four moieties, comprising recognition elementsfor the biological target receptor, plus one tether moiety, primarilyfor control and definition of conformation. These building blocks areassembled together, typically in a sequential fashion, in a second phaseemploying standard chemical transformations. The precursors from theassembly are then cyclized in the third stage to provide the macrocyclicstructures. Finally, a post-cyclization processing stage involvingremoval of protecting groups and optional purification then provides thedesired final compounds (see FIG. 1). This method has been previouslydisclosed in WO 01/25257 and U.S. patent application Ser. No.09/679,331. A general synthetic strategy is shown in FIG. 1.

D. Procedures for the Synthesis of Representative Tethers of the PresentInvention

The important tether component required for compounds of the inventionare synthesized as described in WO01/25257, U.S. Provisional Pat. Appl.Ser. No. 60/491,248 or herein. A standard procedure for the synthesis oftether B is shown in FIG. 2.

-   Step T8-1: Chlorotrimethylsilane (116 mL, 0.91 mol, 1.5 eq) was    added to a suspension of 2-hydroxycinnamic acid (100 g, 0.61 mol,    1.0 eq) in MeOH (500 mL, HPLC grade) over 30 min at 0° C. The    resulting mixture was stirred at rt O/N. The reaction was monitored    by TLC (EtOAc/MeOH: 98/2). Heating the reaction mixture in a hot    water can accelerate the process if necessary. After the reaction    was completed, the reaction mixture was evaporated under reduced    pressure to afford methyl 2-hydroxycinnamate as a white solid    (108.5 g) in quantitative yield. The identity of this intermediate    compound is confirmed by NMR. This reaction can be carried out on    larger (kg) scale with similar results-   Step T8-2: 3,4-Dihydro-2H-pyran (DHP, 140 mL, 1.54 mol, 2.52 eq) was    added dropwise to 2-bromoethanol (108 mL, 1.51 mol, 2.5 eq) in a 2 L    three-neck flask with mechanical stirring at 0° C. over 2 h. The    resulting mixture was stirred for additional 1 h at rt. Methyl    2-hydroxycinnamate from Step T8-1 (108 g, 0.61 mol, 1.0 eq),    potassium carbonate (92.2 g, 0.67 mol, 1-0.1 eq), potassium iodide    (20 g, 0.12 mol, 0.2 eq) and DMF (300 mL, spectrometric grade) were    added to the above flask. The reaction mixture was stirred at 70° C.    (external temperature) for 24 h. The reaction was monitored by TLC    (DCM/Et₂O: 95/5). The reaction was allowed to cool to rt and Et₂O    (450 mL) was added. The inorganic salts were removed by filtration    and washed with Et₂O (3×50 mL). The filtrate was diluted with    hexanes (400 mL) and washed with water (3×500 mL), dried over MgSO₄,    filtered and the filtrate evaporated under reduced pressure. The    crude ester (desired product and excess Br—C₂H₄—OTHP) was used for    the subsequent reduction without further purification.-   Step T8-3: DIBAL (1.525 L, 1.525 mol, 2.5 eq, 1.0 M in DCM) was    added slowly to a solution of the above crude ester from Step T8-2    (0.61 mol based on the theoretical yield) in anhydrous DCM (610 mL)    at −35° C. with mechanical stirring over 1.5 h. The resulting    mixture was stirred for 1.5 h at −35° C., then 1.5 h at 0° C. The    reaction was monitored by TLC (hex/EtOAc: 50/50). When complete,    Na₂SO₄.10H₂O (100 g, 0.5 eq) was slowly added; hydrogen evolution    was observed, when it subsided water was added (100 mL). The mixture    was warmed to rt and stirred for 10 min, then warmed to 40° C. with    hot water and stirred under reflux for 20 min. The mixture was    cooled to rt, diluted with DCM (600 mL), and the upper solution    decanted into a filter. The solid that remained in the flask was    washed with dichloromethane (5×500 mL) with mechanical stirring and    filtered. The filtrate from each wash was checked by TLC, and    additional washes performed if necessary to recover additional    product. In an alternative work-up procedure, after dilution with    DCM (600 mL), the mixture was filtered. The resulting solid was then    continuously extracted with 0.5% TEA in dichloromethane using a    Soxhlet extractor. Higher yield was typically obtained by this    alternative procedure, although it does require more time. The    filtrate was concentrated under reduced pressure and the residue    purified by dry pack (EtOAc/hex/Et₃N: 20/80/0.5) to give the product    alcohol as a yellowish oil (yield: 90%). The identity and purity    were confirmed by NMR.-   Step T8-4: To a mixture of the allylic alcohol from Step T8-3 (28 g,    0.100 mol, 1.0 eq) and collidine (0.110 mol, 1.1 eq) in 200 mL of    anhydrous DMF under N₂ was added anhydrous LiCl (4.26 g, 0.100 mol,    1.0 eq.) dissolved in 100 mL of anhydrous DMF. The mixture was then    cooled to 0° C., and MsCl (12.67 g, 0.110 mol, 1.1 eq., freshly    distilled over P₂O₅), was added dropwise. The reaction was allowed    to warm to rt and monitored by TLC (3:7 EtOAc/hex). When the    reaction was complete, NaN₃ (32.7 g, 0.500 mol, 5.0 eq.) was added.    The reaction mixture was stirred at rt O/N with progress followed by    NMR. When the reaction was complete, the mixture is poured into an    ice-cooled water bath, and extracted with diethyl ether (3×). The    combined organic phases were then washed sequentially with citrate    buffer (2×), saturated sodium bicarbonate (2×), and finally with    brine (1×). The organic layer was dried with MgSO₄, filtered and the    filtrate concentrated under reduced pressure. The allylic azide was    obtained in 90% combined yield, and was of sufficient quality to use    as such for the following step.-   Step T8-5: PPh₃ (25.9 g, 0.099 mol, 1.5 eq) was added at 0° C. to a    solution of the allylic azide from Step T8-4 (20.0 g, 0.066 mol, 1.0    eq.) in 100 mL of THF. The solution was stirred for 30 min at 0° C.    and 20 h at rt. Water (12 mL) was then added and the resulting    solution was heated at 60° C. for 4 h. The solution was cooled to    rt, 2N HCl (15 mL) added and the mixture stirred for 90 min at    50° C. The separated organic phase was extracted with 0.05 N HCl    (2×100 mL). The combined aqueous phase was washed with Et₂O (5×150    mL) and toluene (4×150 mL) (more extraction could be necessary,    follow by TLC), which were combined and back-extracted with 0.05 N    HCl (1×100 mL). This acidic aqueous phase from back-extraction was    combined with the main aqueous phase and washed with ether (5×150    mL) again. The pH of the aqueous phase was then adjusted to 8-9 by    the addition of sodium hydroxide (5 N). Care must be exercised to    not adjust the pH above 9 due to the reaction conditions required by    the next step. The aqueous phase was concentrated under reduced    pressure (aspirator, then oil pump) or lyophilized to dryness.    Toluene (2×) was added to the residue and then also evaporated under    reduced pressure to remove traces of water. The crude product    (desired amino alcohol along with inorganic salt) was used for the    next reaction without further purification.-   Step T8-6: A mixture of the crude amino alcohol from Step T8-5 (0.5    mol based on the theoretical yield), Ddz-OPh (174 g, 0.55 mol, 1.1    eq) and Et₃N (70 mL, 0.5 mol, 1.0 eq) in DMF (180 mL) was stirred    for 24 h at 50° C. Additional DMF is added if required to solubilize    all materials. The reaction was monitored by TLC (hex/EtOAc: 50/50,    ninhydrin detection). After the reaction was complete, the reaction    mixture was diluted with Et₂O (1.5 L) and water (300 mL). The    separated aqueous phase was extracted with Et₂O (2×150 mL). The    combined organic phase was washed with water (3×500 mL) and brine    (1×500 mL), dried over MgSO₄, filtered and the filtrate concentrated    under reduced pressure. The layers were monitored by TLC to ensure    no product was lost into the aqueous layer. If so indicated, perform    one or more additional extractions with Et₂O of the aqueous phase to    recover this material. The crude product was purified by dry pack    (recommended column conditions: EtOAc/hex/Et₃N: 35/65/0.5 to    65/35/0.5) to give the tether Ddz-T8 as a pale yellow syrup (yield:    ˜40%). The identity and purity of the product was confirmed by NMR.

¹H NMR (DMSO-d₆): 1.6 ppm (s, 6H, 2×CH₃), 3.6-3.8 ppm (wide s, 10H,2×OCH₃, 2×OCH₂), 3.95 ppm (triplet, 2H, CH₂N), 6-6.2 ppm (m, 2H, 2×CH),6.2-6.5 ppm (m, 3H, 3×CH, aromatic), 6.6-7.6 ppm (m, 5H, aromatic).

A standard procedure for the synthesis of tether T9 is shown in FIG. 3.

Tether T9 can also be synthesized from T8 by reduction as in step T9-3or with other appropriate hydrogenation catalysts known to those in theart.

A standard procedure for the synthesis of Ddz propargylamine is shown inFIG. 4.

In a dried three-neck flask, a solution of propargylamine (53.7 g, 0.975mol, 1.5 eq) in degassed DMF (Drisolv, 388 mL) was treated with Ddz-N₃(170.9 g, 0.65 mol, 1.0 eq), tetramethylguanidine (TMG, 81.4 mL, 0.65mol, 1.0 eq) and DIPEA (113.1 mL, 0.65 mol, 1.0 eq) and stirred at 50°C. O/N. The reaction was monitored by TLC (conditions: 25/75 EtOAc/hex.R_(f): 0.25; detection: UV, ninhydrin). Upon completion, DMF wasevaporated under reduced pressure until dryness and the residuedissolved in Et₂O (1 L). The organic solution was washed sequentiallywith citrate buffer (pH 4.5, 3×), saturated aqueous sodium bicarbonate(2×), and brine (2×), then dried with MgSO₄, filtered and the filtrateevaporated under reduced pressure. A pale orange solid was obtained.This solid was triturated with 1% EtOAc in hex, then collected byfiltration and dried under vacuum (oil pump) to provide the desiredproduct (153.4 g, 85.2%).

A standard procedure for the synthesis of tether T10 is shown in FIG.5A.

Two alternative routes to this tether have been developed. The firstsynthetic approach proceeded starting from the commercially availablemonobenzoate of resorcinol (T10-0). Mitsunobu reaction under standardconditions with the protected amino alcohol from Example 9, followed bysaponification of the benzoate provided T10-1 in good yield afterrecrystallization. Alkylation of the phenol with 2-bromoethanol usingthe optimized conditions shown permitted the desired product Ddz-T10 tobe obtained after dry pack purification in 42% yield.

A second synthetic route to T10 is shown in FIG. 5B.

From resorcinol, two successive Mitsunobu reactions are conducted withthe appropriate two carbon synthons illustrated, themselves derived from2-aminoethanol and ethylene glycol, respectively, through knownprotection methodologies. Lastly, deprotection of the silyl ether, alsounder standard conditions provided Boc-T10.

Although the yields in the two methods are comparable, the firstrequired less mechanical manipulation and is preferred for largerscales.

A standard procedure for the synthesis of tether T11 is shown in FIG. 6.

A standard procedure for the synthesis of tether T12 is shown in FIG. 7.

In a 3-L flame-dried three-neck flask, a solution of(aminomethyl)phenylthiobenzyl alcohol (12-0, 96 g, 0.39 mol) in degassedDMF (1 L, 0.4 M) was prepared. To this was added DdzN₃ (0.95 eq),followed by TMG (0.39 mol, 49 mL). The reaction was stirred for 10 min,then DIPEA (68 mL, 0.39 mol) added. The mixture was heated at 50° C.under N₂ until TLC indicated no DdzN₃ remained (48 h typically). (TLCeluent: EtOAc:Hex 50:50; detection: ninhydrin). Upon completion, to thereaction mixture was added 3 L citrate buffer and the separated aqueouslayer extracted with Et₂O (3×1500 mL). The combined organic phase waswashed sequentially with citrate buffer (2×200 mL), water (2×200 mL) andbrine (2×200 mL). The organic layer was dried over MgSO₄, filtered andthe filtrate evaporated under reduced pressure. A dark orange oil wasobtained, which was purified by dry-pack. For this procedure, the oilwas first dissolved in EtOAc:Hex:DCM:TEA (20:80:1:0.5, v/v/v/v). At thispoint, a little extra DCM was sometimes required to ensure completedissolution. The solution was loaded onto the column, then the columneluted with EtOAc:Hex:DCM:Et₃N (20:80:1:0.5) until all the impuritieswere separated out as indicated by TLC, paying particular attention tothat closest to the desired product. The elution was then continued withEtOAc:Hex:Et₃N 30:70:0.5 (v/v/v) and finally with EtOAc:hexanes:Et₃N(50:50:0.5) to elute the desired product. After removal of the solventfrom the fractions containing the product under reduced pressure, theresidue was dissolved in the minimum amount of DCM, a three-fold largervolume of hexanes added, then the solvents again evaporated underreduced pressure. This treatment was repeated until an off-white foamwas obtained. The latter solidified while drying under vacuum (oilpump). Alternatively, the material yielded a solid after sequentialconcentration with DCM (1×) and hexanes (2×). Tether Ddz-T12 wasobtained as an off-white solid (85-90% yield).

Example 29 Standard Procedure for Attachment of Tethers Utilizing theMitsunobu Reaction Example 29-A Using PPh₃-DIAD Isolated Adduct

To a 0.2 M solution of the appropriate tether (1.5 eq) in THF orTHF-toluene (1:1) was added the PPh₃-DIAD (pre-formed by mixingequivalent amounts of the reagents and isolated by evaporation ofsolvent, see Example 29-C) adduct (1.0 eq.). The resultant mixture wasmanually agitated for 10 sec (the solution remained turbid), then addedto the resin. Alternatively, the resin was added to the solution. Thereaction suspension was agitated O/N (after ˜5 min the mixture becomeslimpid). The resin was filtered and washed 2×DCM, 1× toluene, 1× EtOH,1× toluene, 1× (DCM/MeOH), 1× (THF/MeOH), 1× (DCM/MeOH), 1× (THF/MeOH),2×DCM, then dried in the standard manner.

Example 29-B Using “PPh₃-DIAD In Situ Procedure”

To a 0.2 M solution of the appropriate tether (4 eq) in THF orTHF-toluene (1:1) was added triphenylphosphine (4 eq). The resultantmixture was manually shaken until a homogenous solution was obtained,then added to the resin. Alternatively, the resin (or IRORI™ MiniKans®(NEXUS Biosystems, Poway, Calif.), miniaturized microreactors,containing resin) was added to the solution. To this suspension was thenadded DIAD (3.9 eq) and the reaction agitated O/N. Note: Since thereaction is exothermic, for larger scales, the reaction should be cooledin an ice bath. In addition, an appropriate vent must be supplied toallow any pressure build-up to be released. The resin was filtered andwashed DCM (2×), toluene (1×), EtOH (1×), toluene (1×), DCM/MeOH (1×),1×THF/MeOH (1×), DCM/MeOH (1×), THF/MeOH (1×), 2×DCM, then dried in thestandard manner.

A procedure for the synthesis of PPh₃-DIAD adduct is shown in FIG. 8.

DIAD (1 eq) was added dropwise to a well-stirred solution oftriphenylphosphine (1 eq) in THF (0.4 M) at 0° C. under nitrogen. Themixture was then maintained at 0° C. with stirring for 30 min. The whitesolid obtained was collected by filtration (use medium sized frittedfilters), washed with cold anhydrous THF until the washes werecolorless, and lastly washed once with anhydrous Et₂O. The white solidproduct was then vacuum-dried (oil pump) and stored under nitrogen.(Note: The PPh₃-DIAD adduct can be made in larger than immediatelyrequired quantity and stored under nitrogen; it is very important tostore this reagent under anhydrous conditions.)

Example 30 Standard Procedure for Attachment of Tethers via ReductiveAmination as Shown in FIG. 9

In certain instances, the Mitsunobu process of Example 29 cannot beapplied or is not efficient for incorporation of the tether. Hence,reductive amination has been developed as an alternative that can beemployed for tether incorporation as illustrated hereinbelow for one ofthe preferred tethers. Similar chemistry can be used to incorporateother tethers of the present invention.

The Tether (30-2) with the amine protected as its Ddz derivative wasefficiently oxidized to the corresponding aldehyde 30-2 using SO₃.pyr inDMSO-Et₃N-DCM. This aldehyde (0.14 mmol, 56 mg, 1.5 eq based uponloading of resin support) was dissolved in a 1:3 mixture of TMOF-MeOH(DriSolv, 4 mL) at rt. To this was added the resin containing thetripeptide (30-1, as its trifluoroacetic acid salt from the deprotectionof the terminal amine), the mixture was agitated briefly to wet theresin, and then borane-pyridine complex (as the commercially available 8M solution, 23 μL, 2 eq) was introduced to the suspension. The reactionwas agitated O/N, then the resin filtered, washed with DCM (2×), THF(1×), DCM/MeOH [3:1] (1×), THF/MeOH [3:1] (1×), DCM (2×) and dried inthe standard manner. Care must be taken to ensure that the desired resinbound product 30-3 is not contaminated with the dialkylated material.However, even if the reaction does not proceed to completion or if asmall amount of the dialkylation side product is present, the materialis of sufficient purity for the macrocyclization reaction.

A standard procedure for the synthesis of tether T28 is shown in FIG.10.

Henry reaction of 2-hydroxybenzaldehyde 28-0 provided 28-1 in 79% yield.This was followed by reduction first with sodium borohydride, then withcatalytic hydrogenation, to give the amine, which was then protected asits Boc derivative, 28-2. Yields of these first two steps were lower onlarger scales. Alkylation of 28-2 with the TBDMS ether of2-bromoethanol, itself synthesized by standard methods, gave 28-3 in 74%yield. Deprotection of the silyl ether under standard conditions yieldedthe desired protected tether, Boc-T28. Alternative use of ethylenecarbonate for the phenol alkylation to avoid the protection/deprotectionsteps, gave 73% yield.

A standard procedure for the synthesis of tether T32 is shown in FIG.11.

A standard procedure for the synthesis of tether T33a and T33b is shownin FIGS. 12A and 12B.

The construction to the (R)-isomer of this tether (T33a) wasaccomplished from 2-iodophenol (33-0) and (S)-methyl lactate (33-A).Mitsunobu reaction of 33-0 and 33-A proceeded with inversion ofconfiguration in excellent yield to give 33-1. Reduction of the ester tothe corresponding alcohol (33-2) also occurred in high yield and wasfollowed by Sonagashira reaction with Ddz-propargylamine. The alkyne inthe resulting coupling product, 33-3, was reduced with catalytichydrogenation. Workup with scavenger resin provided the desired product,Ddz-T33a.

The synthesis of the (S)-enantiomer (Ddz-T33b) was carried out in anidentical manner in comparable yield starting from (R)-methyl lactate(33-B). See FIG. 12B.

Standard procedures for the synthesis of various tethers are shown inthe figures: tether T34 (FIG. 13), tether T35 (FIG. 14), tether T36(FIG. 15), tether T37 (FIG. 16), tether T38 (FIG. 17), tether T39 (FIG.18), tether T40 (FIG. 19), tether T41 (FIG. 20) and tether T42 (FIG.21).

E. Examples of Synthetic Strategies for the Macrocyclic Compounds of theInvention

FIG. 22 presents a scheme depicting a thioester strategy for macrocycliccompounds of the present invention.

It should be noted that one or more of the amino acids indicated can bereplaced by corresponding hydroxy acids and coupled to the next buildingblock utilizing methods known to those in the art.

Example 47 Standard Procedure for Macrocyclization with Thioester Linker

The resin containing the cyclization precursor is combined in anappropriate vessel with pre-washed MP-carbonate resin [ArgonautTechnologies, Foster City, Calif., commercially supplied MP-carbonateresin was treated with 3×THF (1 L per 400 g) and dried O/N at 30° C. ina vacuum oven] (1.4 to 1.6 eq relative to the initial loading of thesynthesis resin). A 0.2 M DIPEA solution in THF was then added to thecombined resins (1 mL/60 mg MP-carbonate resin) and the suspensionagitated O/N at rt. Subsequently, the resin was filtered and rinsed2×THF. The combined filtrates are collected together in an appropriatevessel, then the volatile contents evaporated under reduced pressure [inaddition to the standard methods, solvent can also be removed in vacuousing centrifugal evaporation (ThermoSavant Discovery®, SpeedVac® orcomparable) (Thermo Electron Corporation, Waltham, Mass.)] to providethe crude macrocycles.

Example 48 Standard Procedure for Silver-Assisted Macrocyclization withThioester Linker

Except for the cyclization itself and subsequent work-up, this procedureis identical to that of Example 47. The resin containing the cyclizationprecursor was combined in an appropriate vessel with pre-washedMP-carbonate resin [Argonaut Technologies, commercially suppliedMP-carbonate resin was treated with THF (3×, 1 L per 400 g) and driedO/N at 30° C. in a vacuum oven] (1.4 to 1.6 eq relative to the initialloading of the synthesis resin). To this was added THF (1 mL per 100 mgresin) and silver trifluoroacetate (1 eq relative to the initial loadingof the resin). Finally, an amount of DIPEA sufficient to obtain a 0.2 Msolution was added. The reaction mixture was agitated at rt O/N. Thesolution was then filtered and the resins washed 2×THF. The filtratesare collected together in an appropriate vessel, then evaporated underreduced pressure [(the volatile contents could also be removed in vacuousing centrifugal evaporation (ThermoSavant Discovery®, SpeedVac® orcomparable)] to provide the crude macrocycles. For this procedure,silver trifluoroacetate should be stored in a dessicator between uses.In addition, it is recommended to use a new bottle of THF (or a bottlethat has been recently opened under N₂ or Ar) to minimize formation ofsilver oxide.

Additionally, a ring-closing metathesis (RCM) strategy, as developed byGrubbs et al. can also be used to access some of the macrocycliccompounds of the invention (see for example U.S. Pat. No. 5,811,515;Grubbs, R. H. et al. J. Org. Chem. 2001, 66, 5291-5300; Fürstner, A.Angew. Chem. Int. Ed. 2000, 39, 3012-3043).

To access certain derivatives of compounds of the present invention,additional reactions from those in the general scheme were required. Forsome, it was advantageous to react the functionality to be derivatizedprior to the formation of the macrocyclic ring. The cyclic structure canrestrict access of reagents to that functionality. For example, in thesynthesis of N-methyl and N-acyl derivatives of macrocycles, where thesecondary nitrogen atom of the ring is the site of derivatization, thereaction is preferred to be performed prior to the application of theappropriate cyclization protocol.

In other cases, for example the derivatization of side chainfunctionality, the reaction was best performed after formation of themacrocyclic ring. For example, further reaction of amino moieties onside chains examples was typically efficiently done by reaction of thepartially protected macrocycle. In this manner, acylation,sulfonylation, alkylation (via reductive amination), guanidine and ureaformation were performed via standard methods.

Table 1, hereinbelow, shows a representative, but by no means exclusive,summary of the chemical synthesis of several representative compounds ofthe invention.

TABLE 1 Synthesis of Representative Compounds of the Present InventionTether Additional AA₁ AA₂ AA₃ Tether Attachment Steps 1 Bts-D-Tyr(tBu)Boc-D-Val Boc-Nva Ddz-T8 Example 29 none 2 Bts-D-Phe Boc-D-Val Boc-NvaBoc-T8 Example 29 none 3 Bts-D-Phe Boc-D-Val Boc-Nva Boc-T9 Example 29none 4 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ddz-T9 Example 29 none 5Bts-D-Tyr(tBu) Boc-D-Ala Boc-Nva Ddz-T8 Example 29 none 6 Bts-D-Tyr(tBu)Boc-D-Val Boc-Met Ddz-T8 Example 29 none 7 Bts-D-Tyr(tBu) Boc-D-ValBoc-Nle Ddz-T8 Example 29 none 8 Bts-D-Tyr(tBu) Boc-D-Val Boc-Phe Ddz-T8Example 29 none 9 Bts-D-Tyr(tBu) Boc-D-Val Boc-Val Ddz-T8 Example 29none 10 Bts-D-Tyr(tBu) Boc-D-Val Boc-Leu Ddz-T9 Example 29 none 11Bts-D-2-Nal Boc-D-Val Boc-Nva Boc-T8 Example 29 none 12 Bts-D-Tyr(tBu)Boc-D-Val Boc-Abu Ddz-T8 Example 29 none 13 Bts-D-Phe Boc-D-Val Boc-LeuBoc-T9 Example 29 none 14 Bts-D-2-Nal Boc-D-Val Boc-Leu Boc-T9 Example29 none 15 Bts-D-Phe(3Cl) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 16Bts-D-Phe(4Cl) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 17Bts-D-Trp(Boc) Boc-D-Val Boc-Nva Ddz-T9 Example 29 none 18Bts-D-Tyr(tBu) Boc-D-2-Abu Boc-Nva Ddz-T9 Example 29 none 19Bts-D-Phe(4F) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 20 Bts-D-PheBoc-D-Val Boc-Leu Boc-T8 Example 29 none 21 Bts-D-2-Nal Boc-D-ValBoc-Leu Boc-T8 Example 29 none 22 Bts-D-Tyr(OMe) Boc-D-Val Boc-NvaBoc-T9 Example 29 none 23 Bts-D-1-Nal Boc-D-Val Boc-Nva Boc-T9 Example29 none 24 Bts-D-2-Thi Boc-D-Val Boc-Nva Boc-T9 Example 29 none 25Bts-D-Phe(2Cl) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 26Bts-D-Tyr(tBu) Boc-D-Val Boc-Cpa Ddz-T9 Example 29 none 27 Bts-D-4-ThzBoc-D-Val Boc-Nva Boc-T9 Example 29 none 28 Bts-D-3-Pal Boc-D-ValBoc-Nva Boc-T9 Example 29 none 29 Bts-D-Tyr(tBu) Boc-D-Val Ddz-Dap(Boc)Ddz-T9 Example 29 none 30 Bts-D-Tyr(tBu) Hnva(THP) Boc-Nva Ddz-T9Example 29 none 34 Bts-D-Tyr(tBu) Ddz-D-Tyr(tBu) Boc-Nva Ddz-T8 Example29 None 38 Bts-D-Tyr(tBu) Boc-D-Val Boc-Ala Ddz-T8 Example 29 none 39Bts-D-Tyr(tBu) Boc-D-Val Boc-□-Ala Ddz-T8 Example 29 none 40Bts-D-Tyr(tBu) Boc-D-Val Boc-Gly Ddz-T8 Example 29 none 41Bts-D-Tyr(tBu) Boc-DPhe Boc-Nva Ddz-T8 Example 29 none 52 Bts-D-Tyr(tBu)Boc-D-Val Boc-Phg Ddz-T8 Example 29 none 55 Bts-D-Tyr(tBu) Ddz-D-ValDdz-Lys(Boc) Ddz-T8 Example 29 none 56 Bts-D-Tyr(tBu) Ddz-D-ValDdz-Orn(Boc) Ddz-T8 Example 29 none 57 Bts-D-Tyr(tBu) Ddz-D-ValDdz-Ser(tBu) Ddz-T8 Example 29 none 58 Bts-D-Tyr(tBu) Ddz-D-ValDdz-Tyr(tBu) Ddz-T8 Example 29 none 59 Bts-D-Tyr(tBu) Ddz--D-ValDdz-Trp(Boc) Ddz-T8 Example 29 none 60 Bts-D-Tyr(tBu) Boc-D-ValBoc-Tyr(OMe) Ddz-T8 Example 29 none 65 Bts-D-Tyr(tBu) Boc-D-Val Boc-NvaDdz-T2 Example 29 none 71 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ddz-T10Example 29 none 72 Bts-D-Tyr(tBu) Boc-D-Val Boc-2-Nal Ddz-T8 Example 29none 76 Bts-D-Tyr(tBu) Boc-D-2-Nal Boc-Nva Ddz-T8 Example 29 none 77Bts-D-Tyr(tBu) Boc-D-Nle Boc-Nva Ddz-T8 Example 29 none 80Bts-D-Tyr(tBu) Boc-D-Val Boc-Ile Ddz-T8 Example 29 none 85Bts-D-Tyr(tBu) Boc-D-Val Boc-D-Nva Ddz-T8 Example 29 none 87 Bts-D-BipBoc-D-Val Boc-Nva Boc-T9 Example 29 none 88 Bts-D-Tyr(tBu) Boc-D-ValBoc-Nva Ddz-T9 Example 29 none 89 Bts-D-Hfe Boc-D-Val Boc-Nva Boc-T9Example 29 none 90 Bts-D-Dip Boc-D-Val Boc-Nva Boc-T9 Example 29 none 91Bts-D-Tyr(tBu) Boc-D-Nva Boc-Nva Ddz-T9 Example 29 none 92Bts-D-Tyr(tBu) Boc-D-Tle Boc-Nva Ddz-T9 Example 29 none 96Bts-D-Tyr(tBu) Boc-β-Ala Boc-Nva Ddz-T9 Example 29 none 97Bts-D-Tyr(tBu) Boc-D-Chg Boc-Nva Ddz-T9 Example 29 none 98Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ddz-T18 Example 29 none 99Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ddz-T15 Example 29 none 109Bts-D-Tyr(tBu) Boc-D-Val Ddz-Dab(Boc) Ddz-T9 Example 29 none 110Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ddz-T11 Example 29 none 111Bts-D-Tyr(tBu) Boc-D-Val Hval(THP) Ddz-T9 Example 29 none 112Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ddz-T9 Example 29 none 120Bts-D-Tyr(tBu) Boc-D-Pro Boc-Nva Ddz-T8 Example 29 none 121Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ac-T8-NH2 Example 29 none 122Boc-D-3-Pal Boc-D-Val Boc-Nva Boc-T9 Example 30 none 123 Boc-D-2-PalBoc-D-Val Boc-Nva Boc-T9 Example 30 none 124 Boc-D-4-Pal Boc-D-ValBoc-Nva Boc-T9 Example 30 none 125 Bts-D-Tyr(tBu) Boc-D-Cpg Boc-NvaBoc-T9 Example 29 none 126 Bts-D-Tyr(tBu) Boc-D-Val Boc-NMeLeu Boc-T9Example 29 none 127 Boc-D-His(Mts) Boc-D-Val Boc-Nva Boc-T12 Example 30none 128 Bts-D-Tyr(OMe) Boc-D-Val Boc-Leu Boc-T9 Example 29 none 129Bts-D-1-Nal Boc-D-Val Boc-Leu Boc-T9 Example 29 none 130 Bts-D-2-ThiBoc-D-Val Boc-Leu Boc-T9 Example 29 none 131 Bts-D-Phe(3Cl) Boc-D-ValBoc-Leu Boc-T9 Example 29 none 132 Bts-D-Phe(4Cl) Boc-D-Val Boc-LeuBoc-T9 Example 29 none 133 Bts-D-Phe(4F) Boc-D-Val Boc-Leu Boc-T9Example 29 none 134 Bts-D-Phe(3Cl) Boc-D-Val Boc-Leu Boc-T2 Example 29none 135 Bts-D-Tyr(OMe) Boc-D-Val Boc-Leu Boc-T11 Example 29 none 136Bts-D-1Nal Boc-D-Val Boc-Leu Boc-T11 Example 29 none 137 Bts-D-2-ThiBoc-D-Val Boc-Leu Boc-T11 Example 29 none 138 Bts-D-Phe(3Cl) Boc-D-ValBoc-Leu Boc-T11 Example 29 none 139 Bts-D-Phe(4Cl) Boc-D-Val Boc-LeuBoc-T11 Example 29 none 140 Bts-D-Phe(4F) Boc-D-Val Boc-Leu Boc-T11Example 29 none 141 Bts-D-Tyr(OMe) Boc-D-Val Boc-Cpa Boc-T9 Example 29none 142 Bts-D-1-Nal Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 143Bts-D-2-Thi Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 144 Bts-D-Phe(3Cl)Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 145 Bts-D-Phe(4Cl) Boc-D-ValBoc-Cpa Boc-T9 Example 29 none 146 Bts-D-Phe(4F) Boc-D-Val Boc-CpaBoc-T9 Example 29 none 147 Bts-D-Tyr(OMe) Boc-D-Val Boc-Cpa Boc-T11Example 29 none 148 Bts-D-1-Nal Boc-D-Val Boc-Cpa Boc-T11 Example 29none 149 Bts-D-Phe(3Cl) Boc-D-Val Boc-Cpa Boc-T11 Example 29 none 150Bts-D-Phe(4Cl) Boc-D-Val Boc-Cpa Boc-T11 Example 29 none 151Bts-D-Phe(4F) Boc-D-Val Boc-Cpa Boc-T11 Example 29 none 152Bts-D-Tyr(OMe) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 none 153Bts-D-1-Nal Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 none 154Bts-D-2-Thi Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 none 155Bts-D-Phe(3Cl) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 none 156Bts-D-Phe(4Cl) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 none 157Bts-D-Phe(4F) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 none 158Bts-D-Phe(3Cl) Ddz-D-Val Ddz-Dap(Boc) Ddz-T11 Example 29 none 159Bts-D-Tyr(But) Boc-D-Ile Boc-Nva Boc-T9 Example 29 none 160Bts-D-Tyr(But) Boc-D-allolle Boc-Nva Boc-T9 Example 29 none 161Boc-D-Phe(4CH2NHFmoc) Boc-D-Val Boc-Nva Boc-T9 Example 30 none 162Bts-D-Phe(2Me) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 163Bts-D-Phe(3Me) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 164Bts-D-Phe(4Me) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 165Bts-D-Phe(3OMe) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 166Bts-D-Phe(2OMe) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 167 Bts-D-3-Boc-D-Val Boc-Nva Boc-T9 Example 29 none benzothienyl 168 Bts-D-3-ThiBoc-D-Val Boc-Nva Boc-T9 Example 29 none 169 Bts-D-□-HomoPhe(3Cl)Boc-D-Val Boc-Nva Boc-T9 Example 29 none 170 Bts-D-Phe(3,4diCl)Boc-D-Val Boc-Nva Boc-T9 Example 29 none 171 Bts-D-Phe(3,4diF) Boc-D-ValBoc-Nva Boc-T9 Example 29 none 172 Bts-D-Phe(3,4diOMe) Boc-D-Val Boc-NvaBoc-T9 Example 29 none 173 Bts-D-1Nal Hnva(THP) Boc-Nva Boc-T9 Example29 none 174 Bts-D-Tyr(OMe) Hnva(THP) Boc-Nva Boc-T9 Example 29 none 175Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Boc-T33b Example 29 none 176Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Boc-T33a Example 29 none 177Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Boc-T28 Example 29 none 178Bts-D-Tyr(OMe) Ddz-D-Val Ddz-Ser(tBu) Ddz-T9 Example 29 none 179Bts-D-1-Nal Ddz-D-Val Ddz-Ser(tBu) Ddz-T9 Example 29 none 180Bts-D-2-Thi Ddz-D-Val Ddz-Ser(tBu) Ddz-T9 Example 29 none 181Bts-D-Phe(3Cl) Ddz-D-Val Ddz-Ser(tBu) Ddz-T9 Example 29 none 182Bts-D-Phe(4Cl) Ddz-D-Val Ddz-Ser(tBu) Ddz-T9 Example 29 none 183Bts-D-Phe(4F) Ddz-D-Val Ddz-Ser(tBu) Ddz-T9 Example 29 none 184Bts-D-1-Nal Ddz-D-Val Ddz-Dap(Boc) Ddz-T11 Example 29 none 185Bts-D-Phe(4Cl) Ddz-D-Val Ddz-Dap(Boc) Ddz-T11 Example 29 none 186Ddz-D-Tyr(tBu) Ddz-D-Val Ddz-His(Mts) Ddz-T9 Example 30 none 187Bts-D-Phe(3CF3) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 188Bts-D-Phe(3F) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 189Bts-D-Phe(4NO2) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 190Bts-D-3-benzothienyl Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 191Bts-D-Phe(3OMe) Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 192Bts-D-Phe(3,4diCl) Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 193Bts-D-Phe(3,4diF) Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 194Bts-D-Tyr(OMe) Boc-D-Val Boc-Nva Boc-T34 Example 29 none 195Bts-D-Tyr(OMe) Boc-D-Val Boc-Nva Boc-T38 Example 29 none 196Bts-D-Phe(3Cl) Boc-D-Val Boc-Cpa Ddz-T32(Boc) Example 29 none 197Bts-D-Phe(3Cl) Boc-D-Val Boc-Cpa Boc-T34 Example 29 none 198Bts-D-Phe(3Cl) Boc-D-Val Boc-Cpa Boc-T38 Example 29 none 199Bts-D-Phe(3Cl) Boc-D-Val Boc-Cpa Boc-T41 Example 29 none 200Bts-D-Phe(3Cl) Boc-D-Val Boc-Cpa Boc-T8 Example 29 none 201 Bts-D-1-NalBoc-D-Val Boc-Nva Boc-T8 Example 29 none 202 Bts-D-Phe(3OMe) Boc-D-ValBoc-Nva Boc-T8 Example 29 none 203 Bts-D-Phe(4Cl) Ddz-D-Val Ddz-Dap(Boc)Ddz-T9 Example 29 acetylation 204 Bts-D-Phe(4Cl) Ddz-D-Val Ddz-Dap(Boc)Ddz-T9 Example 29 guanidinylation 205 Bts-D-Phe(3Cl) Boc-D-ValBoc-NMeLeu Boc-T9 Example 29 none 206 Bts-D-Phe(4Cl) Ddz-D-ValDdz-Dap(Boc) Ddz-T9 Example 29 mesylation 207 Bts-D-Phe(4Cl) Ddz-D-ValDdz-Dap(Boc) Ddz-T9 Example 29 TMS- isocyanate followed by dilute acid208 Bts-D-Tyr(tBu) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29guanidinylation 209 Bts-D-Tyr(tBu) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example29 acetylation 210 Bts-D-Tyr(tBu) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example29 reductive amination with acetone 211 Bts-D-Phe(4Cl) Ddz-D-ValDdz-Dap(Boc) Ddz-T9 Example 29 reductive amination with excessformaldehyde 212 Bts-D-Phe(4Cl) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29reductive amination with acetone 213 Bts-D-Tyr(3,5dil) Boc-D-Val Boc-NvaBoc-T9 Example 29 none 214 Bts-D-Tyr(OMe) Boc-D-Val Boc-Hse(Bzl) Boc-T9Example 29 hydrogenolysis for protecting group removal 215Bts-D-Tyr(tBu) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 reductiveamination with excess formaldehyde 216 Bts-D-Phe(3Cl) Boc-D-Val Boc-CpaBoc-T40 Example 29 none 217 Bts-D-Phe(3Cl) Boc-D-Val Boc-Cpa Boc-T36Example 29 none 218 Bts-D-Phe(3Cl) Boc-D-Val Boc-Nva Boc-T39 Example 29none 219 Bts-D-Phe(3Cl) Boc-D-Val Boc-Nva Boc-T37 Example 29 none 220Bts-D-Phe(3Cl) Boc-D-Val Boc-Nva Boc-T39 Example 29 none 221Bts-D-Phe(3Cl) Boc-D-Val Boc-Nva Boc-T35 Example 29 none 222Bts-D-Tyr(3tBu) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 223Bts-D-Tyr(But) Boc-D-Val Boc-Nva Boc-T9 Example 29 acetylation 224Bts-D-1-Nal Boc-D-Val Boc-Leu Boc-T9 Example 29 reductive amination withformaldehyde 225 Bts-D-1-Nal Boc-D-Val Boc-Leu Boc-T9 Example 29acetylation 226 Bts-D-1-Nal Boc-D-Val Boc-Leu Boc-T9 Example 29reductive amination with aldehyde 227 Bts-D-1-Nal Boc-D-Val Boc-LeuBoc-T9 Example 29 reductive amination with benzaldehyde Notes Any aminoacid or tether designated as the Boc derivative could be substitutedwith the corresponding Ddz derivative.D. Analytical Data for Selected Compounds of the Invention

¹H and ¹³C NMR spectra were recorded on a Varian Mercury 300 MHzspectrometer (Varian, Inc., Palo Alto, Calif.) and are referencedinternally with respect to the residual proton signals of the solvent.Information about the conformation of the molecules in solution can bedetermined utilizing appropriate two-dimensional NMR techniques known tothose skilled in the art. HPLC purifications were run on a WatersXterra® MS C18 column, using the Waters FractionLynx® system (WatersCorporation, Milford, Mass.). Automated medium pressure chromatographicpurifications were performed on an Isco CombiFlash® 16× system withdisposable silica or C18 cartridges that permitted up to sixteen (16)samples to be run simultaneously (Teledyne Isco, Inc., Lincoln, Nebr.).MS spectra were recorded on a Waters Micromass® Platform II or ZQ™system. HRMS spectra were recorded with a VG Micromass ZAB-ZFspectrometer. Chemical and biological information were stored andanalyzed utilizing the ActivityBase® database software (ID BusinessSolutions Ltd., Guildford, Surrey, UK).

General Methods for Analytical HPLC Analyses

HPLC analyses are performed on a Waters Alliance® system 2695 running at1 mL/min using an Xterra MS C18 column 4.6×50 mm (3.5 μm). A Waters 996PDA provided UV data for purity assessment (Waters Corporation, Milford,Mass.). An LCPackings (Dionex Corporation, Sunnyvale, Calif.) splitter(50:40:10) allowed the flow to be separated in three parts. The firstpart (50%) went to a Micromass® Platform II MS equipped with an APCIprobe for identity confirmation. The second part (40%) went to anevaporative light scattering detector (ELSD, Polymer Laboratories, nowpart of Varian, Inc., Palo Alto, Calif., PL-ELS-1000™) for purityassessment and the last portion (10%) to a chemiluminescence nitrogendetector (CLND, Antek® Model 8060, Antek Instruments, Houston, Tex.,part of Roper Industries, Inc., Duluth, Ga.) for quantitation and purityassessment. Data was captured and processed utilizing the most recentversion of the Waters Millenium® software package (Milford, Mass.).

An example LC method suitable for compounds of the present inventionuses MeOH as solvent A, H₂O as solvent B and 1% TFA/H₂O as solvent D.Initial mobile-phase composition is 5% A, 85% B and 10% D. Details ofthe standard gradient method are shown below:

Time A % B % D % Curve 0.00 5 85 10 6 1.00 5 85 10 6 6.00 50 40 10 69.00 50 40 10 6 14.00 90 0 10 6 17.00 90 0 10 6 17.50 5 85 10 6 20.00 585 10 6

Compounds 2-6, 8-10, 56, 65 and 144 are as defined in Table (3),hereinbelow.

Compound 2

Yield: 12 mg pure macrocycle was obtained (CLND quantification).

¹H NMR (300 MHz, DMSO-d₆) δ 8.83 (m, 1H); 8.53 (m, 1H); 7.63 (m, 1H);7.4-7.08 (m, 7H); 7.00-6.84 (m, 2H); 6.60 (d, 15 Hz, 1H); 6.41 (dt, 15Hz, 5.4 Hz, 1H); 4.35 (m, 1H); 4.25-4.05 (m, 3H); 3.94 (dt, 1H, 6 Hz, 15Hz); 3.79 (dd, 1H, 3.6 Hz, 8.4 Hz); 3.60 (m, 1H); 3.52-3.40 (bd, 1H);3.22-3.06 (m, 4H); 1.88 (m, 2H); 1.54-1.28 (m, 2H); 1.25 (d, 3H, 4.8Hz); 1.22 (d, 3H, 2.7 Hz); 0.92-0.80 (m, 6H).

HRMS calc. for C₃₀H₄₀N₄O₄: 520.3049. found 520.3057±0.0016

HPLC [standard gradient method (refers to that presented in GeneralMethods for Analytical HPLC Analyses)] t_(R)=9.55 min.

Compound 4

Yield: 12 mg pure macrocycle was obtained (CLND quantification).

¹H NMR (300 MHz, DMSO-d₆) δ 9.35 (b, 1H); 8.98 (b, 1H); 5.52 (d, 1H, 8.4Hz); 8.38 (b, 1H); 7.25 (b, 1H); 7.13-7.07 (m, 4H); 6.86 (t, 2H, 7.5Hz); 6.57 (d, 2H, 8.7 Hz); 4.33 (b, 1H); 4.21-4.02 (m, 3H); 3.78 (dd,1H, 3.3 Hz; 8.1 Hz); 3.65-3.54 (m, 1H); 3.31-3.23 (m, 1H); 3.13-3.02 (m,4H); 2.78-2.2.28-2.18 (m, 1H); 2.0-1.80 (m, 2H); 1.50-1.30 (m, 3H); 1.25(d, 3H, 4.5 Hz); 1.22 (d, 3H, 4.5 Hz); 1.01 (d, 3H, 6.6 Hz); 0.90 (d,3H, 6.6 Hz); (t, 3H, 7.5 Hz).

¹³C NMR (75.5 MHz, DMSO-d₆) δ 172.22; 171.37; 157.77; 157.44; 156.04;131.76; 130.80; 130.70; 127.88; 121.82; 115.83; 111.71; 62.13; 60.62;54.21; 52.81; 47.13; 42.47; 33.31; 29.69; 29.30; 28.61; 20.36; 19.44;18.72; 17.60; 13.97.

HRMS calc. for C₃₀H₄₂N₄O₅: 538.3155. found: 538.3145±0.0016

HPLC (standard gradient) t_(R)=8.12 min.

Compound 5

Yield: 17 mg pure macrocycle was obtained (CLND quantification).

¹H NMR (300 MHz, DMSO-d₆) δ 9.02 (b, 1H); 8.47 (d, 1H, 8.4 Hz); 7.7 (b,1H); 7.58 (d, 1H, 5.4 Hz); 7.28 (dd, 1H, 7.8 Hz, 0.8 Hz); 7.20 (t, 1H,9.0 Hz, 0.8 Hz); 7.14 (d, 2H, 8.4 Hz); 6.98-6.91 (m, 3H); 6.66 (d, 8.7Hz); 6.63 (d, 1H, 15.0 Hz); 6.43 (dt, 1H, 6.0 Hz, 15.0 Hz); 4.28-3.86(m, 6H); 3.60-3.40 (m, 2H); 3.22-3.12 (m, 1H0; 3.05 (d, 2H, 5.4 Hz);1.92-1.80 (m, 1H); 1.56-1.40 (m, 1H); 1.36-1.20 (m, 2H); 1.25 (d, 3H,6.6 Hz); 0.84 (t, 3H, 7.2 Hz).

¹³C NMR (75.5 MHz, DMSO-d₆) δ 172.54; 171.86; 158.97; 158.56; 127.39;155.84; 131.62; 129.73; 129.20; 129.02; 128.43; 126.30; 124.51; 122.01;115.85; 112.88; 61.23; 52.90; 51.23; 47.08; 42.66; 36.13; 33.30; 21.14;19.57; 17.07; 14.14; 11.49.

HRMS calc. for C₂₈H₃₆N₄O₅: 508.2685. found: 508.2681±0.0015

HPLC (standard gradient) t_(R)=7.67 min.

Compound 6

Yield: 16 mg pure macrocycle was obtained (CLND quantification).

¹H NMR (300 MHz, DMSO-d₆) δ 9.37 (b, 1H); 8.87 (b, 1H); 8.61 (d, 1H, 8.7Hz); 7.62 (b, 1H); 7.27 (d, 1H, 7.8 Hz); 7.21 (t, 1H, 8.4 Hz); 7.14 (d,2H, 8.4 Hz); 6.98-6.87 (m, 3H); 6.64 (d, 2H, 8.1 Hz); 6.70 (d, 1H, 15.6Hz); 6.39 (dt, 1H, 6.3 Hz, 15.6 Hz); 4.44-4.36 (m, 1H); 4.34-4.08 (m, 2Hz); 4.45-3.92 (dt, 1H, 6.9 Hz, 15.6 Hz); 3.74 (dd, 1H, 3.6 Hz, 8.4 Hz);3.54-3.26 (m, 3H); 3.22-3.02 (m, 3H); 2.60-2.36 (m, 4H); 2.24-2.14 (m,1H); 2.02 (s, 3H); 1.96-1.89 (m, 1H); 1.80-1.66 (m, 1H); 1.01 (d, 3H,6.3 Hz); 0.90 (d, 3H, 6.6 Hz).

¹³C NMR (75.5 MHz, DMSO-d₆) δ 171.51; 171.26; 158.90; 158.49; 157.38;155.86; 131.63; 129.82; 129.21; 128.86; 128.63; 126.21; 121.98; 115.83;112.83; 62.11; 61.06; 51.97; 47.10; 42.78; 30.91; 30.67; 29.34; 20.37;19.39; 15.06.

HRMS calc. for C₃₀H₄₀N₄O₅S: 568.2719. found: 568.2711±0.0017

HPLC R_(t) (general method) 7.92 min.

Compound 8

Yield: 27 mg pure macrocycle was obtained (CLND quantification).

¹H NMR (300 MHz, DMSO-d₆) δ 9.05 (b, 1H); 8.43 (b, 1H); 8.34 (d, 1H, 9.3Hz); 7.40 (b, 1H); 6.97 (d, 1H, 7.5 Hz); 6.92-6.74 (m, 9H); 6.67-6.54(m, 2H); 6.33-6.25 (m, 3H); 6.10 (dt, 1H, 5.7 Hz, 16.2 Hz); 4.22 (dt,1H, 0.9 Hz, 12 Hz); 3.94-6.66 (m, 4H); 3.30 (dd, 1H, 3.6 Hz, 7.8 Hz);3.24 (m, 1H); 3.18 (m, 1H); 2.85-2.68 (m, 3H); 2.44-2.23 (m, 2H); 1.32(o, 1H, 7.5 Hz); 0.97-0.89 (m, 1H); 0.42 (d, 3H, 6.6 Hz); 0.01 (d, 3H,6.6 Hz).

¹³C NMR (75.5 MHz, DMSO-d₆) δ 171.20; 157.35; 155.88; 139.12; 131.61;130.87; 129.74; 129.21; 128.77; 128.88; 126.85; 126.19; 121.97; 115.82;112.84; 62.04; 61.10; 55.07; 50.01; 47.09; 42.85; 37.42; 29.11.

HRMS calc. For C₃₄H₄₂N₄O₅: 586.3155. found: 586.3145±0.0017

HPLC R_(t) (general method) 9.34 min.

Compound 9

Yield: 17 mg pure macrocycle was obtained (CLND quantification).

¹H NMR (300 MHz, DMSO-d₆) δ 9.39 (b, 1H); 8.83 (b, 1H); 8.29 (d, 1H, 9.3Hz); 7.62 (b, 1H); 7.28 (d, 1H, 6.6 Hz); 7.20 (t, 1H, 6.9 Hz); 7.12 (d,2H, 7.8 Hz); 6.98-6.91 (m, 2H); 6.63 (d, 2H, 8.4 Hz); 6.58 (d, 1H, 16.2Hz); 6.40 (dt, 1H, 5.7 Hz, 16.2 Hz); 4.29-4.13 (m, 3H); 4.03-3.92 (m,2H); 3.52 (m, 1H); 3.15-3.05 (m, 3H); 2.45-2.37 (m, 1H); 1.96-1.88 (m,1H); 1.25 (dd, 2H, 4.5 Hz; 6 Hz); 1.01 (d, 3H, 6.3 Hz); 0.91 (d, 3H, 6.6Hz); 0.86 (d, 3H, 7.2 Hz); 0.81 (d, 3H, 6.6 Hz).

¹³C NMR (75.5 MHz, DMSO-d₆) δ 171.85; 171.17; 157.37; 155.87; 131.59;129.88; 129.18; 128.97; 128.78; 128.51; 126.16; 121.97; 115.83; 112.85;61.55; 61.18; 58.15; 54.22; 47.08; 42.89; 36.32; 29.35; 29.00; 20.34;19.56; 18.73; 17.44.

HRMS calc. for C₃₀H₄₀N₄O₅ 536.2998. found: 536.2990±0.0017.

HPLC (standard gradient) t_(R)=8.15 min.

Compound 10

Yield: 24 mg pure macrocycle was obtained (CLND quantification).

¹H NMR (300 MHz, DMSO-d₆) δ 9.33 (b, 1H); 8.82 (b, 1H); 8.56 (d, 1H, 8.3Hz); 7.60 (b, 1H); 7.27 (d, 2H, 7.8 Hz); 7.20 (t, 1H, 7.8 Hz); 7.13 (d,2H, 8.4 Hz); 6.95 (t, 2H, 7.8 Hz); 6.64 (d, 2H, 8.4 Hz); 6.57 (d, 1H,15.4 Hz); 6.38 (dt, 1H, 15.4 Hz, 5.8 Hz); 4.26-4.10 (m, 3H); 3.96 (dt,1H, 5.4 Hz, 8.4 Hz); 3.77 (dd, 1H, 3.7 Hz, 7.8 Hz); 3.51-3.24 (m, 3H);3.18-3.02 (m, 3H); 1.90 (h, 1H, 6.4 Hz); 1.73-1.54 (m, 2H); 1.45 (dt,1H, 6.7 Hz, 0.9 Hz); 0.99 (d, 3H, 6.6 Hz); 0.89 (d, 3H, 6.3 Hz); 0.87(d, 3H, 6.0 Hz); 0.80 (d, 3H, 6.3 Hz).

¹³C NMR (75.5 MHz, DMSO-d₆) δ 172.23; 171.17; 157.37; 155.88; 131.62;129.82; 129.19; 128.95; 128.59; 126.24; 121.99; 115.84; 112.88; 64.23;61.98; 61.14; 51.43; 61.14; 51.43; 47.07; 42.81; 29.38; 24.85; 24.11;21.00; 20.32; 19.30.

HRMS calc. for C₃₁H₄₂N₄O₅ 550.3155. found: 550.3150±0.0016.

HPLC (standard gradient) t_(R)=8.91 min.

Compound 56

Yield: 16 mg pure macrocycle was obtained (CLND quantification).

¹H NMR (300 MHz, DMSO-d₆) δ 9.39 (b, 1H); 8.90 (b, 1H); 8.67 (d, 1H, 8.4Hz); 7.74 (b, 4H); 7.29-7.08 (m, 4H); 6.99-6.87 (m, 2H); 6.64 (d, 2H,8.1 Hz); 6.61 (d, 1H, 16.5 Hz); 6.40 (dt, 1H, 5.7 Hz, 16.5 Hz);4.40-4.06 (m, 4H); 4.02-3.95 (m, 1H); 3.79 (dd, 1H, 3.6 Hz, 7.8 Hz);3.55-3.30 (m, 2H); 3.16-3.05 (m, 3H); 2.82-2.69 (m, 2H); 2.02-1.85 (m,2H); 1.64-1.43 (m, 3H); 1.29-1.23 (m, 1H); 1.01 (d, 3H, 6.3 Hz); 0.91(d, 3H, 6.3 Hz); 0.86-0.84 (m, 2H).

HPLC (standard gradient) t_(R)=5.71 min.

Compound 65

Yield: 17 mg pure macrocycle was obtained (CLND quantification).

¹H NMR (300 MHz, DMSO-d₆) δ 9.60 (b, 1H); 9.39 (b, 1H); 8.88 (b, 1H);8.70 (d, 1H, 7.5 Hz); 8.57 (d, 1H, 4.2 Hz); 7.27 (t, 6 Hz); 6.96 (d, 2H,8.4 Hz); 6.66 (d, 2H, 8.4 Hz); 5.78-5.68 (m, 1H); 5.42-5.33 (m, 1H);3.96-3.89 (m, 1H); 3.80-3.57 (m, 5H); 3.41-3.34 (m, 1H); 3.10-2.90 (m,1H); 2.78-2.66 (m, 1H); 2.21-2.10 (m, 1H); 2.06-1.93 (m, 1H); 1.70-1.60(m, 1H); 1.52-1.41 (m, 1H); 1.39-1.26 (m, 1H); 1.25 (d, 3H, 4.8 Hz);1.23 (d, 3H, 4.5 Hz); 0.83 (dd, 3H, 3 Hz, 4.5 Hz).

¹³C NMR (75.5 MHz, DMSO-d₆) δ 172.68; 172.63; 159.15; 158.73; 157.38;157.25; 130.89; 124.99; 116.03; 62.51; 62.12; 54.29; 49.27; 42.47;32.77; 30.43; 28.85; 20.46; 19.59; 18.72; 17.39; 13.90; 13.09.

HRMS calc. for C₂₄H₃₆N₄O₄: 444.2736. found: 444.2726±0.0013

HPLC (standard gradient) t_(R)=6.80 min.

Compound 144

¹H NMR (300 MHz, CD₃OD) δ 7.4 (m, 1H); 7.27 (dt, 1H, 1.5 Hz, 6.6 Hz);7.22-7.14 (m, 2H); 7.08-6.98 (m, 2H); 6.78 9t, 2H, 6.6 Hz); 4.45-4.39(m, 2H); 4.15 (d, 2H, 8.1 Hz); 7.74 (d, 1H, 9.3 Hz); 3.54 (d, 1H, 10.8Hz); 3.35-3.22 (m, 2H); 3.20 (q, 1H, 1.5 Hz); 2.82-2.71 (m, 1H);2.61-2.55 (m, 1H); 2.21-2.11 (m, 1h); 2.02-1.94 (m, 1H); 1.74-1.40 (m,5H); 1.04 (d, 3H, 6.6 Hz); 0.93 (d, 3H, 6.6 Hz); 0.74-0.64 9m, 1H);0.45-0.28 (m, 2H); 0.15-0.08 (m, 1H); 0.06-0.02 (m, 1H).

¹³C NMR (75.5 MHz, CD₃OD) δ 173.29; 172.14; 167.51; 155.47; 134.86;134.81; 130.38; 130.31; 128.81; 128.25; 127.44; 121.63; 110.39; 107.71;105.02; 67.10; 66.66; 62.81; 62.06; 60.10; 53.99; 41.44; 36.07; 31.91;30.01; 29.18; 28.94; 27.79; 23.68; 23.15; 19.08; 18.25; 8.17; 4.98;3.16.

HRMS: calc. for C₃₁H₄₁N₄O₄Cl 568.2816. found 568.2802±0.0017

F. Mass Spectral Data for Selected Compounds of the Invention

TABLE 2 Analysis of selected compounds of the invention Molecular WeightMonoisotopic M + H Molecular Formula (calculated) Mass Found 1C30H40N4O5 536.7 536 537 2 C30H40N4O4 520.7 520 521 3 C30H42N4O4 522.7522 523 4 C30H42N4O5 538.7 538 539 5 C28H36N4O5 508.6 508 509 6C30H40N4O5S 568.7 568 569 7 C31H42N4O5 550.7 550 551 8 C34H42N4O5 586.7586 587 9 C30H40N4O5 536.7 536 537 10 C31H42N4O5 550.7 550 551 11C34H44N4O4 572.7 572 573 12 C29H38N4O5 522.6 522 523 13 C31H44N4O4 536.7536 537 14 C35H46N4O4 586.8 586 587 15 C30H41N4O4Cl 557.1 556 557 16C30H41N4O4Cl 557.1 556 557 17 C32H43N5O4 561.7 561 562 18 C29H40N4O5524.7 524 525 19 C30H41N4O4F 540.7 540 541 20 C31H42N4O4 534.7 534 53521 C35H44N4O4 584.7 584 585 22 C31H44N4O5 552.7 552 553 23 C34H44N4O4572.7 572 573 24 C28H40N4O4S 528.7 528 529 25 C30H41N4O4Cl 557.1 556 55726 C31H42N4O5 550.7 550 551 27 C27H39N5O4S 529.7 529 530 28 C29H41N5O4523.7 523 524 29 C28H39N5O5 525.6 525 526 30 C30H41N3O6 539.7 539 540 34C34H40N4O6 600.7 600 601 38 C28H36N4O5 508.6 508 509 39 C28H36N4O5 508.6508 509 40 C27H34N4O5 494.6 494 495 41 C34H40N4O5 584.7 584 585 52C33H38N4O5 570.7 570 571 55 C31H43N5O5 565.7 565 566 56 C30H41N5O5 551.7551 552 57 C28H36N4O6 524.6 524 525 58 C34H40N4O6 600.7 600 601 59C36H41N5O5 623.7 623 624 60 C35H42N4O6 614.7 614 615 65 C24H36N4O4 444.6444 445 71 C29H40N4O6 540.7 540 541 72 C38H42N4O5 634.8 634 635 76C38H42N4O5 634.8 634 635 77 C31H42N4O5 550.7 550 551 80 C31H42N4O5 550.7550 551 85 C30H40N4O5 536.7 536 537 87 C36H46N4O4 598.8 598 599 88C34H50N4O5 594.8 594 595 89 C31H44N4O4 536.7 536 537 90 C36H46N4O4 598.8598 599 91 C30H42N4O5 538.7 538 539 92 C31H44N4O5 552.7 552 553 96C28H38N4O5 510.6 510 511 97 C33H46N4O5 578.7 578 579 98 C24H39N5O4 461.6461 462 99 C24H39N5O4 461.6 461 462 109 C29H41N5O5 539.7 539 540 110C29H41N5O5 539.7 539 540 111 C30H41N3O6 539.7 539 540 112 C31H44N4O5552.7 552 553 120 C30H38N4O5 534.6 534 535 121 C32H45N5O6 595.7 595 596122 C31H43N4O4Cl 571.2 570 571 123 C29H41N5O4 523.7 523 524 124C29H41N5O4 523.7 523 524 125 C30H40N4O5 536.7 536 537 126 C32H46N4O5566.7 566 567 127 C30H38N6O3S 562.7 562 563 128 C32H46N4O5 566.7 566 567129 C35H46N4O4 586.8 586 587 130 C29H42N4O4S 542.7 542 543 131C31H43N4O4Cl 571.2 570 571 132 C31H43N4O4Cl 571.2 570 571 133C31H43N4O4F 554.7 554 555 134 C25H37N4O3Cl 477.0 476 477 135 C31H45N5O5567.7 567 568 136 C34H45N5O4 587.8 587 588 137 C28H41N5O4S 543.7 543 544138 C30H42N5O4Cl 572.1 571 572 139 C30H42N5O4Cl 572.1 571 572 140C30H42N5O4F 555.7 555 556 141 C32H44N4O5 564.7 564 565 142 C35H44N4O4584.7 584 585 143 C29H40N4O4S 540.7 540 541 144 C31H41N4O4Cl 569.1 568569 145 C31H41N4O4Cl 569.1 568 569 146 C31H41N4O4F 552.7 552 553 147C31H43N5O5 565.7 565 566 148 C34H43N5O4 585.7 585 586 149 C30H40N5O4Cl570.1 569 570 150 C30H40N5O4Cl 570.1 569 570 151 C30H40N5O4F 553.7 553554 152 C29H41N5O5 539.7 539 540 153 C32H41N5O4 559.7 559 560 154C26H37N5O4S 515.7 515 516 155 C28H38N5O4Cl 544.1 543 544 156C28H38N5O4Cl 544.1 543 544 157 C28H38N5O4F 527.6 527 528 158C27H37N6O4Cl 545.1 544 545 159 C31H44N4O5 552.7 552 553 160 C31H44N4O5552.7 552 553 161 C31H45N5O4 551.7 551 552 162 C31H44N4O4 536.7 536 537163 C31H44N4O4 536.7 536 537 164 C31H44N4O4 536.7 536 537 165 C31H44N4O5552.7 552 553 166 C31H44N4O5 552.7 552 553 167 C32H42N4O4S 578.8 578 579168 C28H40N4O4S 528.7 528 529 169 C31H43N4O4Cl 571.2 570 571 170C30H40N4O4Cl2 591.6 590 591 171 C30H40N4O4F2 558.7 558 559 172C32H46N4O6 582.7 582 583 173 C34H43N3O5 573.7 573 574 174 C31H43N3O6553.7 553 554 175 C31H44N4O5 552.7 552 553 176 C31H44N4O5 552.7 552 553177 C29H40N4O5 524.7 524 525 178 C29H40N4O6 540.7 540 541 179 C32H40N4O5560.7 560 561 180 C26H36N4O5S 516.7 516 517 181 C28H37N4O5Cl 545.1 544545 182 C28H37N4O5Cl 545.1 544 545 183 C28H37N4O5F 528.6 528 529 184C31H40N6O4 560.7 560 561 185 C27H37N6O4Cl 545.1 544 545 186 C31H40N6O5576.7 576 577 187 C31H41N4O4F3 590.7 590 591 188 C30H41N4O4F 540.7 540541 189 C30H41N5O6 567.7 567 568 190 C33H42N4O4S 590.8 590 591 191C32H44N4O5 564.7 564 565 192 C31H40N4O4Cl2 603.6 602 603 193C31H40N4O4F2 570.7 570 571 194 C32H48N6O6 612.8 612 613 195 C32H46N4O5566.7 566 567 196 C32H43N6O4Cl 611.2 610 611 197 C32H45N6O5Cl 629.2 628629 198 C32H43N4O4Cl 583.2 582 583 199 C27H39N4O6Cl 551.1 550 551 200C31H39N4O4Cl 567.1 566 567 201 C34H42N4O4 570.7 570 571 202 C31H42N4O5550.7 550 551 203 C30H40N5O5Cl 586.1 585 586 204 C29H40N7O4Cl 586.1 585586 205 C32H45N4O4Cl 585.2 584 585 206 C29H40N5O6SCl 622.2 621 622 207C29H39N6O5Cl 587.1 586 587 208 C29H41N7O5 567.7 567 568 209 C30H41N5O6567.7 567 568 210 C31H45N5O5 567.7 567 568 211 C30H42N5O4Cl 572.1 571572 212 C31H44N5O4Cl 586.2 585 586 213 C30H40N4O5I2 790.5 790 791 214C30H42N4O6 554.7 554 555 215 C30H43N5O5 553.7 553 554 216 C32H43N4O4Cl583.2 582 583 217 C31H40N4O4FCl 587.1 586 587 218 C31H43N4O4Cl 571.2 570571 219 C30H40N4O4Cl2 591.6 590 591 220 C31H43N4O4F 554.7 554 555 221C30H40N4O4FCl 575.1 574 575 222 C34H50N4O5 594.8 594 595 223 C32H44N4O6580.7 580 581 224 C36H48N4O4 600.8 600 601 225 C37H48N4O5 628.8 628 629226 C39H49N5O4S 683.9 683 684 227 C42H52N4O4 676.9 676 677 Notes 1.Molecular formulas and molecular weights (MW) are calculatedautomatically from the structure via ActivityBase ® software (IDBS,Guildford, Surrey, UK) or, for MW only, from the freeware programMolecular Weight Calculator v. 6.32 2. M + H obtained from LC-MSanalysis using the General Method as described 3. All analyses conductedon material after preparative HPLC purificationBiological Methods and Results

The compounds of the present invention were evaluated for their abilityto interact at the human motilin receptor utilizing a competitiveradioligand binding assay as described in Method B1. Furthercharacterization of the interaction can be performed utilizing thefunctional assays described in Methods B2, B3 and B4. Some of thesemethods can be conducted, if so desired, in a high throughput manner topermit the simultaneous evaluation of many compounds. Other assays havealso been described that are suitable for HTS, such as that based uponthe stable expression of a synthetic gene for the human motilinreceptor.

Results for the examination of representative compounds of the presentinvention using Method B1 are presented in Table 3. The binding activityis listed as ranges with the following levels: A=0.001-0.10 μM;B=0.10-1.0 μM; C=1.0-10.0 μM. In addition, the assay results of twoadditional compounds using this Method are shown below. As can beobserved, this demonstrates the activity of a representative bicycliccompound of Formula IV of the invention, which resulted fromincorporation of D-proline as the second recognition building block.Significantly, the lack of binding activity obtained with compound 121,which is the linear analogue of compound 1 (K_(i)=level B), illustratesthe critical importance of the cyclic structure to attaining the desiredinteraction.

Competitive binding curves for two representative compounds of theinvention (Compounds 8 and 11) are presented hereinbelow:

For determination of functional significance of the binding, thecompounds are preferably tested in the Aequorin assay as described inMethod B2, although the procedure of Method B3 is also applicable. Ascan be seen from the data presented in Table 4, the representativecompounds examined act as antagonists at the motilin receptor and aredevoid of agonist activity at the concentrations studied. The functionalactivity is listed as ranges with the following levels: A=0.001-0.10 μM;B=0.10-1.0 μM. The higher sensitivity of the assay of Method B2, almost100 times that of Method C, makes it the preferred one for thisassessment. This is evident in the EC₅₀ values obtained in each for thepositive agonist standard, motilin. Additionally, Method B2 measures theactual signaling event, which makes it more relevant to the effect thatis desired, whereas the assay of Method B3 simply measures GTP turnover.

TABLE 4 Demonstration of Antagonist Activity at the Motilin ReceptorAequorin (Method B2)¹ Compound Binding (K_(i)) IC₅₀ 142 A B 149 A B 167A A 168 A A 212 A A Motilin 0.6 not applicable (human, porcine)²¹Activity is listed as ranges with the following levels: A = 0.001-0.10μM; B = 0.10-1.0 μM ²Human and porcine motilin are the same peptide.

In addition, a common and scientifically-accepted ex vivo assay for themeasurement of agonist or antagonist activity at the motilin receptor isthe contraction of rabbit duodenum or other gastrointestinal smoothmuscle tissue.^(A2-A4) Agonists are defined as compounds thatinduce >50% contraction relative to the motilin peptide, whereasantagonists are defined as compounds that cause >50% inhibition of theresponse to motilin. Compounds of the present invention have shownsignificant antagonist activity in this assay. For example, compound 144exhibited a pA₂=6.95, while compound 165 had a pA₂=7.17, as calculatedfrom the Schild plots of the response obtained at various concentrationsas described in Method B4.

Gastric motility is generally measured in the clinical setting as thetime required for gastric emptying and subsequent transit time throughthe GI tract. Gastric emptying scans are well known to those skilled inthe art an, briefly, comprise use of an oral contrast agent, such asbarium, or a radiolabeled meal. Solid and liquids can be measuredindependently. A test food or liquid is radiolabeled with an isotope(^(99m)Tc) and after ingestion or administration, transit time throughthe GI tract and gastric emptying are measured by visualization usinggamma cameras. These studies are performed before and after theadministration of the therapeutic agent to quantify the efficacy of thecompound.

Example Method B1 Competitive Radioligand Binding Assay (MotilinReceptor)

Materials:

-   -   Membranes were prepared from CHO cells stably transfected with        the human motilin receptor and utilized at a quantity of 1.5        μg/assay point. [PerkinElmer™ SignalScreen®) Product #6110544,        PerkinElmer, Inc., Wellesley, Mass.]    -   [¹²⁵I]-Motilin (PerkinElmer, #NEX-378); final concentration:        0.04-0.06 nM    -   Motilin (Bachem™, #H-4385, Bachem Bioscience Inc., King of        Prussia, Pa.); final concentration: 1 μM    -   Multiscreen® Harvest plates-GF/B (Millipore™, #MAHFB1H60,        Billerica, Mass.)    -   Deep-well polypropylene titer plate (Beckman Coulter™, #267006,        Fullerton, Calif.)    -   TopSeal-A™ (PerkinElmer, #6005185, Wellesley, Mass.)    -   Bottom seal (Millipore™, #MATAH0P00, Billerica, Mass.)    -   MicroScint-0™ (PerkinElmer, #6013611, Wellesley, Mass.)    -   Binding Buffer: 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 1 mM EDTA,        0.1% BSA        Assay Volumes:    -   150 μL of membranes diluted in binding buffer    -   10 μL of compound diluted in binding buffer    -   10 μL of radioligand ([¹²⁵I]-Motilin) diluted in binding buffer        Final Test Concentrations (N=11) for Compounds:    -   10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005 μM.        Compound Handling:

Compounds were provided frozen on dry ice at a stock concentration of 10mM diluted in 100% DMSO and stored at −20° C. until the day of testing.On the test day, compounds were allowed to thaw at room temperature andthan diluted in assay buffer according to the desired testconcentrations. Under these conditions, the maximum final DMSOconcentration in the assay was 0.5%.

Assay Protocol:

In deep-well plates, diluted cell membranes (1.5 μg/mL) are combinedwith 10 μL of either binding buffer (total binding, N=5), 1 μM motilin(non-specific binding, N=3) or the appropriate concentration of testcompound. The reaction is initiated by addition of 10 μl of[¹²⁵I]-motilin (final conc. 0.04-0.06 nM) to each well. Plates aresealed with TopSeal-A, vortexed gently and incubated at room temperaturefor 2 hours. The reaction is arrested by filtering samples throughpre-soaked (0.3% polyethyleneimine, 2 h) Multiscreen Harvest platesusing a Tomtec® Harvester (Tomtec, Hamden, Conn.)), washed 9 times with500 μL of cold 50 mM Tris-HCl (pH 7.4), and than plates are air-dried ina fumehood for 30 minutes. A bottom seal is applied to the plates priorto the addition of 25 μL of MicroScint-0™ to each well. Plates are thensealed with TopSeal-A® and counted for 30 sec per well on a TopCount®Microplate Scintillation and Luminescence Counter (PerkinElmer,Wellesley, Mass.) where results are expressed as counts per minute(cpm).

Data are analyzed by GraphPad™ Prism (GraphPad Software, San Diego,Calif.) using a variable slope non-linear regression analysis. K_(i)values were calculated using a K_(d) value of 0.16 nM for [¹²⁵I]-motilin(previously determined during membrane characterization).

$D_{\max} = {1 - {\frac{\begin{matrix}{{{test}\mspace{14mu}{concentration}\mspace{14mu}{with}\mspace{14mu}{maximal}\mspace{14mu}{displacement}} -} \\{{non}\text{-}{specific}\mspace{14mu}{binding}}\end{matrix}}{{{total}\mspace{14mu}{binding}}\; - \;{{non}\text{-}{specific}\mspace{14mu}{binding}}} \times 100}}$where total and non-specific binding represent the cpm obtained in theabsence or presence of 1 μM motilin, respectively.

Example Method B2 Aequorin Functional Assay (Motilin Receptor)

Materials:

-   -   Membranes were prepared using AequoScreen™ (EUROSCREEN, Belgium)        cell lines expressing the human motilin receptor (cell line        ES-380-A; receptor accession #AF034632). This cell line is        constructed by transfection of the human motilin receptor into        CHO-K1 cells co-expressing G_(α16) and the mitochondrially        targeted Aequorin (Ref #ES-WT-A5).    -   Motilin (Bachem™, #H-4385, Bachem Bioscience Inc., King of        Prussia, Pa.)    -   Assay buffer: DMEM-F12 (Dulbeccoe's Modified Eagles Medium) with        15 mM HEPES and 0.1% BSA (pH 7.0)    -   Coelenterazine (Molecular Probes™, Leiden, The Netherlands)        Final Test Concentrations (N=5) for Compounds:    -   10, 3.16, 1, 0.316, 0.1 μM.        Compound Handling:

Compounds were provided as dry films at a quantity of approximately 1.2mmol in pre-formatted 96-well plates. Compounds were dissolved in 100%DMSO at a concentration of 10 mM and stored at −20° C. until furtheruse. Daughter plates were prepared at a concentration of 500 μM in 30%DMSO with 0.1% BSA and stored at −20° C. until testing. On the test day,compounds were allowed to thaw at room temperature and than diluted inassay buffer according to the desired test concentrations. Under theseconditions, the maximum final DMSO concentration in the assay was 0.6%.

Cell Preparation:

Cells are collected from culture plates with Ca²⁺ and Mg²⁺-freephosphate buffered saline (PBS) supplemented with 5 mM EDTA, pelletedfor 2 minutes at 1000×g, resuspended in assay buffer (see above) at adensity of 5×10⁶ cells/mL and incubated overnight in the presence of 5μM coelenterazine. After loading, cells were diluted with assay bufferto a concentration of 5×10⁵ cells/mL.

Assay Protocol:

For agonist testing, 50 μl of the cell suspension was mixed with 50 μlof the appropriate concentration of test compound or motilin (referenceagonist) in 96-well plates (duplicate samples). The emission of lightresulting from receptor activation was recorded using the FunctionalDrug Screening System 6000 ‘FDSS 6000’ (Hamamatsu Photonics K.K.,Japan).

For antagonist testing, an approximate EC80 concentration of motilin(i.e. 0.5 nM; 100 μL) was injected onto the cell suspension containingthe test compounds (duplicate samples) 15-30 minutes after the end ofagonist testing and the consequent emission of light resulting fromreceptor activation was measured as described in the paragraph above.

Results are expressed as Relative Light Units (RLU). Concentrationresponse curves were analyzed using GraphPad™ Prism® (GraphPad Software,San Diego, Calif.) by non-linear regression analysis (sigmoidaldose-response) based on the equation E=E_(max)/(1+EC₅₀/C)n where E isthe measured RLU value at a given agonist concentration (C), E_(max) isthe maximal response, EC₅₀ is the concentration producing 50%stimulation and n is the slope index. For agonist testing, results foreach concentration of test compound were expressed as percent activationrelative to the signal induced by motilin at a concentration equal tothe EC₈₀ (i.e. 0.5 nM). For antagonist testing, results for eachconcentration of test compound were expressed as percent inhibitionrelative to the signal induced by motilin at a concentration equal tothe EC₈₀ (i.e. 0.5 nM).

Example Method B3 FlashPlate® Motilin [³⁵S]-GTPγS Functional Assay

Materials:

-   -   Membranes were prepared from CHO cells stably transfected with        the human motilin receptor and utilized at a quantity of 1.5        μg/assay point.    -   [PerkinElmer™ SignalScreen® Product #6110544, PerkinElmer, Inc.        Wellesley, Mass.]    -   GTPγS Guanosine 5′-[γ-thio]triphosphate tetralithium salt        (Sigma, #G-8634, Sigma-Aldrich, St. Louis, Mo.)    -   [³⁵S]-GTPγS (PerkinElmer, #NEX-030H)    -   Motilin (Bachem™, #H-4385, Bachem Bioscience Inc., King of        Prussia, Pa.)    -   96-well FlashPlate® white polystyrene microplates (PerkinElmer,        #SMP200, Wellesley, Mass.)    -   Deep-well polypropylene titer plate (Beckman Coulter™, #267006,        Fullerton, Calif.)    -   TopSeal-A™ (PerkinElmer, #6005185, Wellesley, Mass.)    -   Assay Buffer: 50 mM Tris (pH 7.4), 100 mM NaCl, 10 mM MgCl₂, 1        mM EDTA, 1 μM GDP, 0.1% BSA        Assay Volumes:    -   25 μL of compound diluted in assay buffer    -   25 μL of assay buffer (agonist assay) or 0.6 μM motilin (0.1 μM        final concentration) diluted in assay buffer (antagonist assay)    -   100 μL of [³⁵S]-GTPγS diluted in assay buffer        Final Test Concentrations (N=12) for Compounds:    -   50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01 μM.        Compound Handling:

Compounds were provided frozen on dry ice at a stock concentration of 10mM diluted in 100% DMSO and stored at −20° C. until the day of testing.On the test day, compounds were allowed to thaw at room temperature andthan diluted in assay buffer according to the desired testconcentrations. Under these conditions, the maximum final DMSOconcentration in the assay was 0.5%.

Assay Protocol:

CHO membranes were immobilized into 96-well FlashPlate® microplates.Test compound, GTPγS, motilin and [³⁵S]-GTPγS were combined in each wellaccording to the Assay Volumes described above.

For the assay to measure agonist activity, an additional 25 μl of bufferwas added to each well in addition to 25 μL of either buffer (basalvalue, N=4), 1 μM (final conc.) motilin (E_(max) value, N=3), 25 μM(final conc.) GTPγS (non-specific value, N=4), or the appropriateconcentration of test compound (N=3).

For the assay to measure antagonist activity, an additional 25 μL ofeither buffer (unstimulated control) or motilin (0.1 μM final conc.) isadded to each well, in addition to either 25 μL of buffer (basal value,N=3), 1 μM (final conc.) motilin (E_(max) value, N=3), 25 μM (finalconc.) GTPγS (non-specific value, N=4), or the appropriate concentrationof test compound (N=3).

The reaction is initiated by addition of 100 mL of [³⁵S]-GTPγS to eachwell. Each plate is sealed (TopSeal-A™) and incubated in the dark atroom temperature for 150 min. Then, plates are counted for 30 secondsper well on the TopCount® NXT.

Data were analyzed by GraphPad™ Prism® 3.0 (GraphPad Software, SanDiego, Calif.) using non-linear regression analysis (sigmoidaldose-response) for the calculation of IC₅₀/EC₅₀ values.

${{E_{\max}({agonist})}\mspace{14mu}{or}\mspace{14mu}{D_{\max}({antagonist})}} = {\frac{{Top} - {Bottom}}{Bottom} \times 100}$

Where Top and Bottom correspond to the top and bottom values of thedose-response curve calculated by GraphPad™ Prism®).

Example Method B4 Rabbit Duodenum Contractility Assay

Duodenal segments were vertically suspended in organ chambers of 10 mLfilled with Krebs buffer and connected to an isotonic force transducer,with a preload of 1 g. After a stabilization period, the muscle stripswere challenged with 10⁻⁴ M acetylcholine and washed. This was repeateduntil a stable maximal contraction was obtained (2-3 times), with aninterval of at least 20 minutes.

After a stable base line was reached, test compounds were added to thebath. After 15 min incubation, a dose response to motilin was recordedby adding logarithmically increasing concentrations of motilin to thebath (final concentration 10⁻⁹ to 10⁻⁶ M). A blank experiment (no testcompound present) was also performed. At the end of the dose responsecurve, a supramaximal dose of acetylcholine (10⁻⁴ M) was given and thisresponse was used as a reference (100% contraction).

The results of experiments at different concentrations of test compoundwere combined and analyzed to derive the pA₂ value from the Schild plot.

It is appreciated that although specific experimental methods have beendescribed herein for the purposes of illustration, various modificationsto these experimental methods as well as alternate methods ofexperimentation may be used without departing from the scope of thisinvention.

TABLE 3 Binding activity of selected compounds R₁ R₃ R₆ 1

2

3

4

5

CH3

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

34

38

CH₃ 39

H 40

H 41

52

55

56

57

58

59

60

65

71

72

76

77

80

85

H 87

88

89

90

91

92

96

H

97

98

99

109

110

111

112

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

T K_(i) ^(1,2) 1

B 2

A 3

B 4

A 5

B 6

B 7

B 8

B 9

B 10

A 11

A 12

B 13

B 14

B 15

A 16

A 17

B 18

B 19

A 20

B 21

A 22

A 23

A 24

A 25

B 26

A 27

B 28

B 29

B 30

B 34

B 38

C 39

B 40

C 41

C 52

B 55

B 56

B 57

B 58

B 59

B 60

C 65

B 71

B 72

B 76

C 77

C 80

B 85

B 87

B 88

C 89

C 90

C 91

C 92

B 96

C 97

C 98

C 99

C 109

B 110

B 111

B 112

B 122

B 123

B 124

B 125

B 126

B 127

B 128

B 129

A 130

B 131

A 132

A 133

A 134

C 135

B 136

B 137

B 138

B 139

B 140

B 141

A 142

A 143

B 144

A 145

A 146

A 147

B 148

B 149

A 150

B 151

B 152

B 153

B 154

B 155

A 156

A 157

B 158

A 159

B 160

B 161

B 162

B 163

A 164

B 165

A 166

B 167

A 168

A 169

B 170

A 171

A 172

A 173

B 174

B 175

B 176

B 177

B 178

B 179

B 180

B 181

A 182

A 183

B 184

B 185

B 186

B 187

A 188

A 189

B 190

A 191

A 192

A 193

A 194

B 195

A 196

197

198

A 199

B 200

A 201

B 202

A 203

B 204

A 205

B 206

B 207

B 208

B 209

C 210

211

A 212

A 213

B 214

B 215

B 216

A 217

B 218

A 219

B 220

A 221

B 222

A 223

C 224

B 225

B 226

C 227

B Notes Radioligand competitive binding assays performed using Method B1Values reported as ranges: A = 0.001-0.100 μM; B = 0.100-1.0 μM; C =1.0-10.0 μM

X is NH except for:

Compound 223 and 225, X is:

Compound 224, X is NMe

Compound 226, X is:

Compound 227, X is

Z₁, Z₂ and Z₃ are NH except for compounds 30, 173 and 174 and where Z1is O and compound 111 where Z₂ is O.

R₂, R₄ and R₅ are hydrogen except for compound 85 where it is:

m, n₁ and p are zero.

What is claimed is:
 1. A process for preparing a compound of formula(I):

wherein R₁ is C₁-C₄ alkyl; the process comprising: (a) contacting acompound of formula (A):

with a compound of formula (B):

in the presence of an azodicarboxylate reagent and a phosphine reagent,or in the presence of a combined Mitsunobu reagent, to form a compoundof formula (C):

wherein X is halogen or triflate and PG₂ is an ester protecting group;(b) contacting the compound of formula (C) with a reducing agent to forma compound of formula (D):

(c) contacting the compound of formula (D) with a compound of formula(E):

in the presence of a palladium catalyst, optionally a copper salt and/oroptionally an organic base to form a compound of formula (F):

wherein PG₁ is hydrogen or an amine protecting group; (d) contacting thecompound of formula (F) with hydrogen in the presence of a metalcatalyst to form the compound of formula (G):

(e) removing the PG₁ protecting group, when present, from the compoundof formula (G) to provide the compound of formula (I).
 2. The process ofclaim 1, wherein the compound of formula (I) is:


3. The process of claim 1 wherein the compound of formula (B) isselected from the group consisting of:

wherein PG₂ is an ester protecting group.
 4. The process of claim 1,wherein X is iodine.
 5. The process of claim 1, wherein PG₁ is hydrogen.6. The process of claim 1, wherein PG₁ is a carbamate protecting group.7. The process of claim 1, wherein PG₁ is selected from the groupconsisting of tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz),9-fluorenylmethoxycarbonyl (Fmoc),α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz) and allyloxycarbonyl(Alloc).
 8. The process of claim 1, wherein PG₂ is an alkyl group or analkyl group substituted with an aryl group.
 9. The process of claim 1,wherein PG₂ is methyl, ethyl or benzyl.
 10. The process of claim 1,wherein the azodicarboxylate reagent is selected from the groupconsisting of diethylazodicarboxylate (DEAD) anddiisopropylazodicarboxylate (DIAD).
 11. The process of claim 1, whereinthe phosphine reagent is selected from the group consisting oftriphenylphosphine and tributylphosphine.
 12. The process of claim 1,wherein the combined Mitsunobu reagent is atriphenylphosphine-diisopropylazodicarboxylate (DIAD) adduct.
 13. Theprocess of claim 1, wherein the reducing agent is selected from thegroup consisting of an aluminum hydride and a borohydride.
 14. Theprocess of claim 1, wherein the reducing agent is selected from thegroup consisting of diisobutylaluminum hydride (DIBAL-H), lithiumaluminum hydride (LAH), and lithium borohydride.
 15. The process ofclaim 1, wherein the palladium catalyst is selected from the groupconsisting of dichlorobis(triphenylphosphine)palladium(II),dichlorobis(acetonitrile)-palladium(II),dichlorobis(benzonitrile)palladium(II),tetrakis(triphenyl-phosphine)palladium(0) andtris(dibenzylideneacetone)dipalladium(0).
 16. The process of claim 1,wherein the copper salt is a copper halide.
 17. The process of claim 1,wherein the copper salt is copper (I) iodide.
 18. The process of claim1, wherein the copper salt is not present.
 19. The process of claim 1,wherein the organic base is selected from the group consisting of adialkylamine, a trialkylamine and an aromatic amine.
 20. The process ofclaim 1, wherein the organic base is triethylamine (TEA),diisopropylamine or N,N-diisopropylethylamine (DIPEA).
 21. The processof claim 1, wherein the organic base is not present.
 22. The process ofclaim 1, wherein (c) is conducted in the presence of a phosphine. 23.The process of claim 1, wherein the metal catalyst is palladium oncarbon or platinum oxide.
 24. The process of claim 1, wherein compound(G) is subjected to a purification step comprising chromatography or drypack chromatography.
 25. The process of claim 1, wherein contacting thecompound of formula (A) with a compound of formula (B) in the presenceof an azodicarboxylate reagent and a phosphine reagent, or in thepresence of a combined Mitsunobu reagent, to form a compound of formula(C) is replaced by contacting the compound of formula (A) with acompound of formula (G):

wherein Y is a leaving group, R₁ is C₁-C₄ alkyl and PG₇ is an esterprotecting group, in the presence of a base to form the compound offormula (C).
 26. The process of claim 25, wherein Y is a halogen or asulfonate.
 27. The process of claim 25, wherein Y is selected from thegroup consisting of 4-methylbenzenesulfonate (tosylate),methanesulfonate (mesylate), 2-nitrobenezenesulfonate,4-nitrobenezenesulfonate (nosylate), 2,4-dinitrobenezenesulfonate,4-bromobenezenesulfonate (brosylate), trifluoromethanesulfonate(triflate), chloride, bromide and iodide.
 28. The process of claim 25,wherein the base is selected from the group consisting of a hydrogencarbonate salt, a carbonate salt, a trialkylamine and an aromatic amine.